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Chapter 1: Chemical Basis of Life

Four Major Biomolecules

I. Amino Acids: contain an amino group and a carboxylic group and a side chain/R group
 Under physiological conditions, ionized to NH3+ and COO-
 Ex. Alanine and Asparagine
II. Carbohydrates: (monosaccharides/sugars)
 Formula = (CH2O)n
 Ex. Glucose
III. Nucleotides: five-carbon sugar, nitrogen-containing ring, and one or more phosphate groups.
 Ex. Adenosine Triphosphate (ATP)
IV. Lipids: poorly soluble in water due to its bulky hydrocarbon-like structure.
 Ex. Palmitate and Cholesterol

3 Major Biological Polymers

I. Proteins (Polypeptides): Polymers of amino acids.
 Amino acids linked via peptide bonds. (red arrow
signifies peptide bond)
 Can perform tasks such as mediating chemical
reactions and provide structural support.

II. Nucleic Acids (Polynucleotides): polymers of nucleotides; DNA and RNA

 Primary Role: carriers of genetic information, contained in their
sequence of nucleotide residues rather than their 3D shape.
 RNA: nucleotides – adenine, cytosine, guanine, and uracil (ACGU)
 DNA: nucleotides – adenine, cytosine, guanine, and thymine (ACGT)

III. Polysaccharides: contain only one or a few different type of

monosaccharide residue
 Limited potential for carrying genetic information in the sequence of
their residues or for adopting a large variety of shapes and mediating chemical reactions
 Serve as fuel-storage molecules and provide structural support.
 Linked via glycosidic bonds
Energy and Metabolism
 1st Law of Thermodynamics: Energy cannot be created or destroyed.
 Free Energy (Gibbs Free Energy, G, units: J*mol-1)
o Enthalpy (H): heat content of the system
o Entropy (S): measure of how the energy is dispersed within that system; measure of the system’s

G is less than zero for a spontaneous process.

 For a process to occur, the overall change in free energy (G) must be negative.
o A reaction with G<0 (negative) has a decrease in enthalpy and an increase in entropy is
spontaneous at all temperatures.
o A nonspontaneous or endergonic reaction has a free energy change greater than zero; the reverse
reaction is spontaneous.
Thermodynamic spontaneity does NOT indicate how fast a reaction will occur, but if it WILL*
*Dependent on molecules, temperature, or presence of catalyst.

 At equilibrium
o A reaction with a decrease in enthalpy and an increase in entropy is spontaneous at all
 2 law: energy tends to spread out

o Enthalpy increasing while entropy decreasing is NOT possible

o If enthalpy and entropy both increase or both decrease during a reaction, the value of G then
depends on the temperature which governs whether T S term is greater or less than the H

Life is thermodynamically possible

 In order to exist, life must be thermodynamically spontaneous.
o Many of the cells metabolic reactions have free energy changes that are less than zero but some
reactions do not.
o The nonspontaneous reactions are able to proceed in vivo (in a living organism) because they occur
in concert with other reactions that are thermodynamically favorable (coupling).

 Reduction, the gain of electrons is accomplished by the addition of hydrogen or the removal of oxygen.
 The plant or animal that eats can then break down the monosaccharide to use it as fuel to power
other metabolic activities.

In the process the carbon is oxidized it loses electrons through the addition of oxygen or the removal of hydrogen
and ultimately becomes CO2.
 The oxidation of carbon is thermodynamically favorable so it can be coupled to energy requiring
processes such as the synthesis of building blocks and their polymerization to form macromolecules
 Mostly all metabolic processes occur with the aid of catalysts called enzymes which most are proteins
 An organism with its high level of organization represents a state of low energy relative to its
surroundings. (high organization/stability= low energy)
 the organism can maintain this thermodynamically unfavorable state as long as it continually obtains
free energy from its food when it exhausts food, G= 0 resulting in death
The Origin and Evolution of Life
 the ability to replicate is one of the universal characteristics of living organisms
 cells will carry out a set of instructions but will evolve throughout time

 The prebiotic world

o H2, H2O, NH3, and CH4 could have given rise to simple biomolecules such as amino acids, when
sparked by lighting.
o HCN, HCOH, and phosphate could have been converted to nucleotides
o Charles Darwin “warm little pond theory”
Similar molecular building blocks could have accumulated and formed larger structures,
particularly in shallow waters where evaporation would have had a concentrating effect.

The first biological building blocks would have had to polymerize by gaining the capacity for self-replication.

Complement- mirror image copy

Origins of modern cells

o Replicating molecules chances of decreasing depend on natural selection
o This favors a replicator that was chemically stable and had a ready supply of building blocks and free
energy for making copies of itself

CO2 + H2O  (CH2O) + O2

The first cells were able to fix CO2 that is convert it to reduced organic compounds using free energy
released in the oxidation of readily available inorganic compounds such as H2S or Fe2+. Later on
photosynthetic organisms similar to present day cyanobacteria used the suns energy to fix CO 2

 Prokaryotes: small unicellular organisms that lack a discrete nucleus and usually contain no internal
membrane systems.
o This group is composed of two groups Archaea and eubacteria
 Eukaryotes: cells usually larger than prokaryotic cells and contain a nucleus and other membrane bounded
cellular compartments. May be unicellular or multicellular.
o This group called the eukarya includes microscopic organisms as well as familiar macroscopic
plants and animals
o Eukaryotic cells exhibit characteristics exhibit characteristics of both bacteria and archaea
o Eukaryotic cells also contain organelles that are almost certainly the descendants of free living

 The number of sequence differences between two groups of organisms indicates how long ago they
diverged from a common ancestor.
 Species with similar sequences have a longer shared evolutionary history than species with dissimilar
Chapter 2: Aqueous Chemistry
Water Molecules form Hydrogen Bonds
 Water is 60% by mass in humans

 Characteristics of water
o Tetrahedral Bent Geometry
o Polar; Partial negative and positive charges in oxygen and hydrogen
o In Ice  forms lattices, bonds with 4 H2O molecules
o In water  each bond is short, molecules flicker
o Many biomolecules have atoms (hydroxyl, carbonyl, amine, amide) that can Hydrogen Bond with

 Water is highly cohesive

o Accounts for its high surface tension
o Explains why water remains a liquid

Ionic Interactions (Electrostatic Forces in water)

 Intermediate in strength
o Hydrogen Bonds
 In DNA
 In Proteins
o Van-der-waals forces: between polar molecules
o London-dispersion: between non-polar molecules

Why do some drugs contain fluorine?

 Introduction of fluorine allowed alteration to drug’s chemical/biological properties without significantly
altering the shape.
 Fluorine can take place of hydrogen
o High electronegativity, behaves more like Oxygen than hydrogen
 Transforms C-H group to an electron-withdrawing polar C-F group, decreasing basicity.
o Fewer positive charges allow a drug to pass more easily through the membrane
o C-F bond can participate in hydrogen bonding/dipole-dipole interactions
 Better binding = more effective at lower concentrations and fewer side effects.

Water dissolves many compounds

 Water has a high dielectric constant
o Solvent’s ability to diminish the electrostatic attractions between dissolved ions
o Higher dielectric constant, less able the ions to associate with each other.
The hydrophobic effect: Why is it thermodynamically unfavorable to dissolve a
hydrophobic substance in water?
 Enthalpy is needed to break the hydrogen bonds in water to create a hole for non-polar molecules to fit.
o The free energy barrier in solvation is more dependent on entropy
o When a hydrophobic molecule is hydrated, a layer of water molecules that cannot participate in
hydrogen bonding and align themselves so the polar ends are not oriented toward the nonpolar
solute.  Loss of entropy in the system
 Non-polar molecules clump/aggregate in water because they are driven out by unfavorable entropy cost of
individual hydration

 Hydrophobic Effect: exclusion of nonpolar substances

from an aqueous solution
o Do not experience any additional attractive

Amphiphilic molecules experience both hydrophilic interactions and the hydrophobic effect
Amphiphilic molecules: have both hydrophobic and hydrophilic portions
 Ex. Nonpolar hydrocarbon tail and a polar carboxylate head
 Polar groups of amphiphilic molecules are oriented towards the polar solvent and are hydrated, nonpolar
groups aggregate due to the hydrophobic effect to form a spherical micelle.
 Can form a sheet to form bilayers

Hydrophobic Core of lipid bilayer is a barrier to diffusion

 Lipid bilayers close up to form a vesicle
o Traps a volume of aqueous solution, polar solutes remain bc they cannot pass through the
hydrophobic interior of the bilayer (too high of energetic cost)
o Bilayers can prevent diffusion of high concentrated substances to lower concentration (movement
down a concentration gradient, increase of entropy)

Sweat, Exercise and Sport Drinks

 Heat due to metabolic activity can be lost by the vaporization of water, which has a significant cooling effect.
o Increase in skin temperature triggers activity of sweat glands to secrete a solution of Na +, K+, and Cl-
 Exercise at high temperatures can cause fluid loss (up to 2L per hour)
o Fluid loss >2% of body’s weight can impair cardiovascular function
 Fluid intake should match losses due to sweat; rate of intake should be equal with rate of sweating
o For activities less than 90 minutes, water is ok
 Sports Drinks have high carbohydrate content, but only advantageous during prolonged sustained activity
(ex. marathon)
 Normal Diets usually contain enough Na+ and Cl- to match losses in sweat
Acid-Base Chemistry
 Equilibrium constant for Ionization of water
o 2H2 O ⇌ H+ + OH−
o Kw (ionization constant for water) = [H+][OH-] = 10-14
 Has no units
 Defining pH
o Acidic Solutions: [H+] > 10-7
o Basic Solutions: [H+] < 10-7

A pK value describes an acid’s tendency to ionize

 Ka, Acid Dissociation Constant =
 pK = -log (Ka)
 Larger an acid’s Ka, the smaller its pK and the greater its strength

 Henderson-Hasselbach equation: relates pH of a solution to the pK of an acid and the concentration of the
acid (HA) and its conjugate Base (A-)
pH = pK + log
 pK and pH convey protonation state
o At pH < 3.5, amino acids are protonated
 Net Positive Charge (+1)
 COOH and NH3+
o At 3.5 < pH < 9.0, carboxyl is deprotonated
 Charge is neutral (0)
 Zwitter Ions
o At pH > 9.0, both groups are deprotonated
 Net negative charge (-1)
 COO- and NH2
 Rule of thumb: pH = pK +/- 1
o When [A] = 10[HA]
 pH = pK + log (10) = pK + 1
o When [HA] = 10 [A-]
 pH = pK log (0.1) = pK – 1

 When HCl is added to a weak acid in equilibrium with its conjugate base, pH does not change dramatically
because some added protons combine with the conjugate to reform the acid and will not contribute to
increase [H+]
 Effective buffering capacity: generally within one pH unit of its (pK = +/- pH)
Chapter 4 Protein Structure
 Protein: consists of one or more polypeptides (chains of polymerized amino acids)
 Amino Acid: small molecule with an amino (NH3+), a carboxylate (COO-), and R-group
o R group (side chain): determines biological reactivity

20 Amino Acids
 Hydrophobic Amino Acids (NON POLAR)
o Alanine (Ala, A) o Leucine (Leu, L)
o Valine (Val, V) o Isoleucine (Ile, I)
o Phenylalanine (Phe, F) o Methionine (Met, M)
o Tryptophan (Trp, W) o Proline (Pro, P)
o Glycine (Gly, G) (*only aa that is not chiral)
*NOTE: The more surface area, the more nonpolar

 Polar Amino Acids

o Serine (Ser, S) o Asparagine (Asn, N)
o Threonine (Thr, T) o Glutamine (Gln, Q)
o Tyrosine (Tyr, Y) o Histidine (His, H)
o Cysteine (Cys, C)
*NOTE: look for presence of electron-withdrawing atoms (S, N, O); often found in active site of enzymes
because they can facilitate chemical catalysis

 Charged Amino Acids

o Positive/Basic o Negative/Acid
 Histidine (His, H)  Aspartate (Asp, D)
 Arginine (Arg, R)  Glutamate (Glu, E)
 Lysine (Lys, K)

(Refer to flashcards and/or sheet for structures and more information)

 Amino Acids are linked via condensation reaction (water molecule is eliminated)
o Peptide bonds: linkage of two amino acids to form a polypeptide
o Residues: amino acids within the peptide
o Can be broken/hydrolyzed via exo or endopeptidases

 The chain read from the N-terminus to the C-terminus

o N-terminus has free amino group
o C-terminus has free carboxyl gr
Sample Calculation. Estimate net charge of the polypeptide chain: Ala-Arg-Val-His-Asp-Gln
1. At pH = 7.4

2. At pH = 5.0

Sample problem. Draw the structure of the peptide Gly-Lys in its fully protonated forms. Which ionic form
predominates at (a) pH = 1, (b) pH = 7, (c) pH = 13

Four Levels of Protein Structure

I. Primary Structure: sequence of amino acid residues
a. Joined together by peptide bonds (very strong covalent bonds)
II. Secondary Structure: localized conformation of the polypeptide backbone
a. Start to form H bonds between backbone atoms
III. Tertiary Structure: 3-D structure of an entire polypeptide, with all its side chains
a. Final fold of the protein; held together by a combination of H bonds, ionic bonds, disulfide
bonds, van der waals forces
IV. Quaternary Structure: spatial arrangement of polypeptide chains in a protein with multiple subunits
a. True for only the proteins that have more than one polypeptide chain
b. Held together by stronger forces: combinations of ionic bones, van der waals forces, and
disulfide bonds
Primary Structure: sequence of amino acid residues

Secondary Structure: localized conformation of the polypeptide backbone

 Rearrangement of atoms attached in backbone of protein forms repeating structural units that are
maintained by H-bonds contributed to atoms in the main backbone
 Restricted rotation of peptide bonds due to the two resonance forms of the peptide bond
o Groups involved in peptide bonds are strongly polar.
o Rotation around C-C and N-C determines secondary structures of proteins
 The polypeptide chain folds to satisfy as many of the hydrogen-bonding requirements as possible.
o Must adopt a conformation that minimizes steric strain
 Two kinds of secondary structures
I. -Helix: twisted backbone conformation
II. -Sheet: contains multiple polypeptide strands

These are both regular secondary structures: component residues have backbone conformations
that are the same from one residue to the next

-Helix: exhibits a twisted backbone conformation

 Rod-like tightly coiled structure
 H-bonds form between carbonyl oxygen and amino hydrogen
 H-bonds are parallel to the main axis
 3.6 residues per turn
-Pleated Sheets: extended chains of polypeptide sheets arranged in two ways
 Parallel -Pleated Sheets: chains run in the same
 Anti-Parallel -Pleated Sheets: chains run in
opposite directions
 Single sheet may have 2 to more than 12
polypeptide strands

 Irregular secondary structures (loops and coils)

o Loops that link  strands or  helices have residues with irregular secondary structures
o The polypeptide DOES NOT adopt a defined secondary structure (doesn’t mean disordered)
o In every protein, elements of the secondary structure are linked together by polypeptide loops.

 Proteins can have a combination of secondary structures

o Combination: usually the tertiary structure

Tertiary Protein Structure: 3D shape of entire polypeptide and spatial arrangement of all its side
 Overall folding of the peptide backbone, includes its regular and irregular secondary structures
o X-Ray Crystallography: determined protein structure (ex. myoglobin)
o Globular Proteins

 Types of interactions that maintain tertiary structures

I. Hydrophobic Interactions
II. Hydrogen Bonding
III. Disulfide Bridges
IV. Ionic bonds

Proteins have hydrophobic cores

 Globular proteins have at least 2 layers of secondary structures; protein has a definite surface and core
o Protein has a hydrophilic surface and a hydrophobic core
o The greater a residue’s hydrophobicity, the more likely it is in the protein interior.
 Ion Pair: when a charged residue is in the protein interior, it is located near another residue with an
opposite charge for them to interact electrostatically

 Domain: polypeptide segment that has folded into a single structural unit with a hydrophobic core

A two domain protein

Protein structure stabilization via Hydrophobic Effect

 The largest force governing protein structure: Hydrophobic Effect
o Driven by the increase in entropy of surrounding water molecules
o Hydrophobic side chains in interior of protein  Stabilizes the folded
polypeptide backbone

 Hydrogen bonding (by itself) is NOT a major determinant of protein stability

o Helps by fine-tuning the folded conformation that is stabilized by the
hydrophobic effect

Cross-links help stabilize proteins

I. Ion Pairs
II. Disulfide bonds
III. Inorganic ions (Zinc Fingers)

Ion Pairs: form between oppositely charged side chains or N- and C- terminal groups
 Strong electrostatic interaction
 But doesn’t contribute much to protein stability; favorable free energy of electrostatic interaction is offset
by loss of entropy when side chains are fixed.

Disulfide Bonds: form within and between polypeptide chains

 Not essential for stabilizing proteins
 More plentiful in extracellular (oxidizing) environments, prevent protein unfolding under relatively harsh
extracellular conditions

Zinc Fingers: structures of 20-60 residues with one or two Zn2+ ions
o Common in DNA-binding proteins
o Ideal ion for stabilizing proteins; can interact with ligands (S,N, or O) provided by amino acids and
only has one oxidation state
Protein Folding: begins with formation of secondary structures
 In cell, a newly synthesized polypeptides folds as soon as it emerges from the ribosome; part of the
polypeptide may adopt its mature tertiary structure before entire chain is synthesized
o Denatured: chemically unfolded
 Addition of highly soluble substances (ex. salts or urea) that interfere with
the hydrophobic effect causing unfolding of the proteins
o Renatured: refolding

Protein renaturation is NOT random; approaches the native structure (most stable tertiary
structure) through alternate pathways
 Small elements of secondary structure form first then come together due to the
hydrophobic effect to produce a mass with a hydrophobic core; small rearrangements yield
the native structure
The information required for protein folding is all contained in the amino acid

Molecular chaperones are smaller proteins that assist in protein folding.

Proteins undergo processing before reaching mature forms

 Removal of amino acid residues
 Covalent attachment of a group (lipid, carbohydrate, or phosphate) which has a discrete biological
function and helps stabilize the folded conformation of a protein
 Association with metal ions/specific organic molecules

Proteins can adopt more than one conformation

 Minor movement  bending/stretching of individual bonds is required for biological function
 Intrinsically Unstructured proteins (protein segments): highly flexible extended segments rich in
hydrophilic amino acids
 The flexibility of a protein’s conformation has two stable alternatives in dynamic equilibrium that can tip
between different conformations
o Change in cellular conditions (pH or oxidation state)
o Presence of a binding partner

Protein Misfolding and Disease

 Degradation of a protein to its component amino acids is a mechanism in which cells can deal with
misfolded proteins.
 Cystic fibrosis: mutated for of the CFTR protein folds incorrectly and never reaches cellular destination
 Diseases resulting from aggregated misfolded proteins (fibrous deposits - long, insoluble fibers) are
deadliest when in the brain
o Ex. Diseases characterized by fibrous deposits: Alzheimer’s, Parkinson’s and transmissible
spongiform encephalopathies; lead to neurological abnormalitiy and loss of neurons
o Amyloid deposits: aggregated proteins

Alzheimer’s Disease (neurodegenerative): accompanied by intracellular tangles and extracellular plaques

 Tau: fibrous protein material inside cell involved in assembly of microtubules
 Excess amyloid- linked to Alzheimer’s disease
 Early stages of amyloid- misfolding and aggregation are toxic to neurons; ultimate cause
Parkinson’s disease: neurons in a portion of the brain accumulate -synuclein protein
 -synuclein: forms alpha helices upon binding to other molecules; contributes to propensity to form
amloid deposits
 Mutations in the gene for alpha-synuclein lead to increased expression or promote self-aggregation.

Transmissible Spongiform Encephalopathies (TSE) (or Mad Cow Disease)

 Infectious agent is a protein called the prion
 Scrapie form of prion (PrPSC)

Quaternary Structure: spatial arrangement of polypeptide chains in a protein with multiple subunits
 Two or more subunits oriented in 3D space
o Due to noncovalent interactions
 Subunits vs. Domains
o Domains: single chain that can form a local 3D structure; folded polypeptide segments
 Independent structural unit with a hydrophobic core
o Subunit: two or more separate chains in a protein that orient in 3D space to give quaternary
Domain Subunits

 Subunits can be identical are named homodimers, homotetramers, etc.

o Nonidentical chains have a prefix of hetero-
 There are similar forces that hold subunits together in tertiary structures
o Hydrogen bonds; ion pairs; and disulfide bonds

 These interactions give an opportunity for subunits to influence each other’s behavior/cooperativity;
regulates functions not possible in single-subunit proteins

 Advantages of multisubunit protein structure:

o Construct extremely large proteins through incremental addition of small building blocks
o Decreases the impact of errors in transcription and translation

Tools and Techniques for Analyzing Protein Structure

Chromatography: takes advantage of a polypeptides unique properties
 Proteins or other solutes pass through the column at different rates, depending on how they react with
the stationary phase (polar; porous matrix).
 Size-Exclusion Chromatography (gel filtration chromatography)
o Stationary phase has tiny beads with pores of characteristic size.
o Larger proteins are excluded and pass through the column faster
than smaller proteins.
 Ion-Exchange Chromatography
o Exploiting a protein’s net charge at a particular pH.
o Solid phase can have positively charged diethylaminoethyl (DEAE) or negatively charged
carboxymethyl groups (CM)
 Negatively charged proteins bind to DEAE
 Postively charged proteins pass through column
o Anionic groups become protonated

Isoelectric point (pI): pH at which a protein carries no net charge

= ½ (pK1 + pK2)

 Affinity chromatography: separation via the basis of high affinity of the protein of interest for specific
chemical groups
o Takes advantage of unique ability to interact with another molecule
o Ex. Isolation of Concanavalin A, which binds to glucose
 Column has glucose to bind to ConA
 High-performance liquid chromatography (HPLC): carried out in closed columns under high pressure

 Standard approach to sequencing proteins

I. Protein sample is purified
II. Chains are separated
III. Large polypeptides are broken into smaller pieces for individual sequencing
IV. Sequencing (Edman’s Degradation): N-terminus is chemically derivatized, cleaved off and identified
V. Reconstruct sequence; different set of fragments that overlap first is generated

 Mass spectrometry: more efficient; measures the sizes of peptide fragments

o Determines the sequence of amino acids in a polypeptide

Protein structures determined by X-ray crystallography, electron crystallography and NMR spectroscopy
 X-Ray crystallography: performed on samples of proteins that have been crystallized
o X-ray’s bombard and electrons in crystals scatter the x-rays to produce a diffraction pattern
o Trace the polypeptide backbone and discern general shapes
o Crystallized proteins retain some ability to move
 Electron Crystallography: electron beams probe the protein’s structure
o Proteins do not have to be crystallized
o 3D structures can be reconstructed

 Nuclear Magnetic Resonance (NMR) spectroscopy: takes advantage of atomic nuclei (hydrogen) to
resonate in an applied magnetic field
o Spectrum consists of peaks that are analyzed to reveal distances between two H atoms close or
covalently connected to other atoms in space
o Constructs a 3D model of the protein
Chapter 5: Protein Function
 Proteins have various functions:
I. Transport: myoglobin transports oxygen throughout muscles; hemoglobin transports oxygen in
II. Structural: actin forms microfilaments in cells; tubulin dimers constitute microtubules; keratin
filamines in animal hair; collagen is a major protein in connective tissue
III. Motor function: myosin interacts with actin to facilitate muscular movement
IV. Catalysis
V. Immunity
VI. Regulation of Gene Expression

Why focus on myoglobin and hemoglobin?

o Oxygen transport is critical for life
o Hemoglobin mutation can lead to disease
o Characteristic of oxygen binding to myoglobin and hemoglobin are highly observed

Myoglobin: transports oxygen throughout muscles

 Classical globular, tetrameric protein that lacks  structure entirely
 Has an iron-containing porphyrin derivative known as heme which is a
prosthetic group
o Prosthetic group: an organic compound that allows a protein to carry out
a function that a polypeptide cannot perform alone

Heme is a porphyrin that

chelates oxygen transpor

Myoglobin transports oxygen via Iron (Fe) in heme

Oxygen binding to myoglobin depends on oxygen concentration

𝐵𝑜𝑢𝑛𝑑 𝑀𝑏 [𝑀𝑏𝑂2]
Y= = [𝑀𝑏]+[𝑀𝑏𝑂2]
(fractional saturation)
𝑇𝑜𝑡𝑎𝑙 𝑀𝑏

Kd (p50) = [O2] at which myoglobin is half saturated

Kd pO2, partial pressure of O2 (in torr)

The amount of O2 bound to myoglobin (Y) is a function of the

oxygen concentration (pO2) and the affinity of Mb for O2 (K)

O2 binding to the heme group of myoglobin exhibits a hyperbolic curve

Myoglobin and hemoglobin are related by evolution
 Hemoglobin is a heterotetramer which have two alpha and two beta chains, called globin.
o Globins are homologous proteins that evolved from a common evolutionary origin/ancestor
through genetic mutation

Hemoglobin and myoglobin have similar secondary and tertiary structures

 Have a heme group in a hydrophobic pocket
 Have His F8 that ligands the Fe2+ (Ferrous form) – reduced form that binds oxygen
 Have His E7 that forms hydrogen bonds with oxygen

Primary structures not similar (~18% similarity in primary sequence)

Oxygen binds cooperatively to Hemoglobin

Myoglobin O2 binding = Hyperbola

Hemoglobin O2 binding = Sigmoid

* Sigmoidal data indicates cooperativity

Hemoglobin has a lower affinity for O2

 Hemoglobin binding is sigmoidal because at low [O2], hemoglobin is reluctant to bind to the first
O2, but as the concentration (pO2) increases, O2 binding sharply increases
 Indicates that the four heme groups in Hb are not independent and communicate cooperatively

 Cooperative binding: binding of one molecule of O2 to hemoglobin causes a conformational change to

hemoglobin which enhances the ability to bind more O2 molecules
o Exhibits allosteric interactions
o Has more than one binding site

Key physiological function: hemoglobin’s low oxygen affinity and cooperative binding behavior
 In lungs, the pO2 is 100 torr – hemoglobin is 95% saturated with O2
 In tissues, pO2 is 20-40 torr – hemoglobin is 55% saturation; Hb affinity decreases
o Here, O2 released by hemoglobin is readily taken up by myoglobin in muscle cells, since
myoglobin’s affinity for O2 is much higher at relatively low oxygen concentration.

(from lecture)
 At 15 kPa (lungs) Hb 95% saturation - low ability to deliver/donate O2 at high [ ]

 At 5 kPa (tissues) Hb 75% saturation – high ability to donate O2 (low affinity)

o Myoglobin highly saturated in muscle cells

 At 1-2 kPa (exercise) Hb 10% saturation – easier for Hb to deliver O2; myoglobin is source of O2
Erythropoietin (EPO) boosts red blood cell production
 Synthesized by kidneys; individuals with kidney disease are deficient in EPO and develop anemia
o Treatment w recombinant EPO can increase red blood cell production
 Can be abused by endurance sport athletes (like Lance Armstrong….smh)

Conformational shift explains myoglobin’s cooperative behavior

 Shift in conformation between oxy and deoxy states involves rotation of one  unit
o Oxygen binding decreases size of the central cavity

There are two quaternary structures of hemoglobin:

I. Deoxyhemoglobin (T, tense conformation): hemoglobin without any bound O2
 Heme is dome shaped (nonplanar) in deoxygenated Hb; has five ligands
 Reluctant to bind the first O2 molecule because the T conformation is unfavorable for O2 binding

II. Oxyhemoglobin (R, relaxed conformation): hemoglobin with bound O2

 Has 6 ligands
 The following O2 molecules bind with higher affinity due to the R conformation, favorable for O2 binding.

 Allosteric proteins: proteins

with multiple binding sites
o Binding of a small
molecule (ligand) to
one site alters the
ligand-binding affinity
of other sites

pH effects on Hemoglobin Affinity

 At higher pH – hemoglobin saturation is 75%
 At lower pH – hemoglobin saturation is 50% (decreased affinity)
Bohr Effect: reduction of hemoglobin’s oxygen-binding affinity when the pH decreases
 Groups become more acidic and release H+ when O2 binds to protein
 In vivo, tissues release CO2 which enters RBC’s and is converted into bicarbonate (HCO3-)
 In lungs, high oxygen concentration promotes O2 binding to hemoglobin causing release of protons
that combine with bicarbonate to reform CO2 as it’s breathed out

Effect of 2,3 BPG on oxygen binding

*BPG decreases hemoglobin’s O2 affinity

Red blood cells use a mechanism with 2,3 Biphosphogycerate

 BPG binds in central cavity of a deoxygenated (T) hemoglobin
 Five negative charges in BPG interact with positively charged groups in deoxyhemoglobin
 Presence of BPG stabilizes the deoxy conformation of hemoglobin; would bind O2 too tight without

Fetal hemoglobin has a higher O2 affinity than adult hemoglobin, which helps O2 transfer for maternal
circulation across the placenta to the fetus.
Chapter 6 How Enzymes Work
What is an enzyme?
 There are three ways to increase the rate of a chemical reaction:
1. Increase the temperature (adding energy in the form of heat)
2. Increasing the concentrations of the reactants
3. Addition of a catalyst
 Living systems use enzymes
 Ribozymes are catalysts made of RNA (exception)

 Chymotrypsin: a digestive protein that is synthesized in the pancreas and secreted into the small intestine
to break down dietary protein
 Hydrolysis of polypeptides take place in a cleft between the two domains near the side chains of
three residues (His, Asp, Ser) called the active site.
 Catalyzes hydrolysis of peptide bonds at 190 per second
o Rate enhancement of 108 to 1012.
 Acts under mild conditions
 Has a broad range of substrate specificity
o Catalyzes hydrolysis of peptide, amide, and ester bonds following Phe, Trp, and Try.
o P-nitrophenylacetate (an ester) is readily hydrolyzed by chymotrypsin.

 Enzymes have high reaction specificity.

 Most are highly specific for their reactants (substrates) and products.
 Function groups in the active site of an enzyme can distinguish its substrates.

 The activities of enzymes are regulated in order to respond to changing conditions of follow genetically
determined developmental programs.

Enzymes are usually named after the reaction they catalyzed

 All biochemical reactions involve the addition of some substance to another, or its removal, or the
rearrangement of that substance.

Enzyme Classification
I. Oxidoreductases = Oxidation-reduction reactions
II. Transferases = Transfer of functional groups
III. Hydrolases = Hydrolysis reactions
IV. Lyases = Group elimination to form double bonds
V. Isomerases = Isomerization reactions
VI. Ligases = Bond formation coupled with ATP hydrolysis

 Isozymes: multiple enzymes catalyzing the same reaction

How do enzymes differ from nonbiological catalysts?
Explain why an enzyme’s common name may not reveal its biological role.
Chemistry of Catalysis
 In a biochemical reactions, reactants must come together and undergo electronic rearrangement to form

Free energy of Activation (G++): energy-

requiring step of the reaction/energy

Transition state: point of highest energy;

intermediate between reactants and
products. Process of breaking old bonds
and forming new bonds.

 The height of the activation energy

barrier determines the rate of reaction.
 The higher the barrier, the less likely and slower the reaction will occur.

A catalyst provides a reaction pathway with a lower activation energy barrier

 A catalyst decreases the activation energy barrier (G++) by interacting with the reacting molecules so
they are more likely to assume the transition state.
 Doesn’t alter the net free energy change; only provides a pathway from reactants to products through
a transition state that has lower free energy.

 Overall: An enzyme lowers the height of the activation energy barrier (G++) by lowering the energy of the
transition state.

Enzymes use chemical catalytic mechanisms

Cofactors: a small organic molecule/metal ion
that is required for catalytic activity

Coenzymes: cofactors that are organic

molecules, derived from vitamins

 Prosthetic group: a tightly bound coenzyme

that remains in the active site between reactions.
There are three basic chemical catalytic mechanisms used by enzymes:
I. Acid-Base Catalysis: a proton is transferred between the enzyme and substrate
 Acid Catalysis: donation of proton from catalyst – H+ transfer from acid by enzyme lowers free energy of
transition state Amino acids in A-B Catalysis

 Base Catalysis: H+ is abstracted by a base to lower the free energy

of the transition state

II. Covalent Catalysis (Nucleophilic Catalysis): a covalent bond forms between the catalyst and the
substrate during formation of the transition state
 An electron rich group (nucleophile) in the enzyme active site forms a covalent adduct with a
 Undergoes a two-part reaction process, so there is two energy barriers.
 Ser, Tyr, Cys, Lys, and His are often good covalent catalysts

Ex. Catalysis of acetoacetate to acetone via a primary amine (RNH2) to form an imine (Schiff base)
III. Metal Ion Catalysis: A zinc ion stabilizes a developing negative on the transition state
o Metal ions participate in enzymatic reactions by mediating oxidation-reduction reactions
o Or by promoting the reactivity of other groups in the enzyme’s active site through
electrostatic effects.
 A protein-bound metal ion can also interact directly with the reacting substrate.

o Coordinates covalent bonds that hold the

substrate in proper orientation

The catalytic triad of chymotrypsin promotes peptide bond hydrolysis

Chymotrypsin uses both acid-base catalysis and covalent catalysis.
 Dependent on three active site residues: His 57, Asp 102, Ser 195 – Catalytic Triad
 Identified using chemical labeling

Chymotrypsin is a serine protease.

 Ser 195 is essential for catalysis.
 The substrates scissile bond (bond cleaved by hydrolysis) is near Ser 195 when substrate binds to the
Catalytic Mechanism of Chymotrypsin *just know examples*
I. Peptide substrate enters chymotrypsin active site; scissile bond is close to
oxygen of Ser 195
o Removal of Ser hydroxyl proton by His 57 (base catalyst)
o Resulting nucleophilic oxygen (covalent catalyst) attacks carbonyl on

II. Tetrahedral intermediate decomposes

 His 57 is now an acid catalyst, donates proton to nitrogen of
scissile bond, cleaving the bond
 Asp 102 stabilizes His 57 through H-bonding
 Energetically favorable because electron flow is easy

III. Portion of protein is gone; other part is covalently attached to Ser 195
 C-term of cleaved peptide leaves N-term of the substrate (acyl
group) linked to enzyme
 Newly exposed N-terminus

IV. Water enters active site – donates proton to His 57

 His 57 is now a base catalyst
 Hydroxyl group attacks carbonyl of substrate

V. Second tetrahedral intermediate (His 57 – now acid catalyst)

 Donates proton to Ser oxygen
 Causes intermediate to collapse

VI. Enzyme is regenerated; rest of protein is released

 N-terminal portion of the original substrate has a new C-
terminus and diffuses away
 Regenerates the enzyme
Unique properties of Enzyme Catalyst
 Catalytic activity depends on transition state stabilization, proximity effects, induced fit, and electrostatic

Enzymes stabilize the transition state by binding tightly to the transition state.
 Noncovalent interactions form and release free energy, lowering G++

Lock and Key Model

Emil Fisher: catalytic residues must be precisely aligned in the active site

Linus Pauling: an enzyme increases the reaction rate by binding tightly to

the reaction state (not to substrates)
 Substrates strained towards structures of products

Stabilization (tight binding) of transition state occurs in addition to A-B, covalent, or metal ion catalysis.
 Accomplished through close complementarity in shape and charge between active site and transition state

Induced Fit Model (Daniel Koshland)

 When binding to substrates, enzymes undergo pronounced conformational changes to almost fully enclose

Transition State Model

 Not only looks at the active site that recognizes and binds the substrate but orients
the substrate to active it and go through the transition state
 The active site orients the substrate in a way to activate toward reaction
 When the substrate is held in the active site, it takes characteristics of the
transition state

 The catalyst enhances the formation and stabilization of the highly

energetic transition state.

Proximity and Orientation Effects

Enzymes increase reaction rates by bringing reactive groups into close proximity to increase frequency of
 When substrates bind to enzyme, they are brought into correct orientation by freezing out the
translational and rotational motions
Induced Fit
Ex. The binding of hexokinase to glucose causes a
conformational change in the enzyme to fit the substrate

Electrostatic catalysis
: Non-aqueous active site allows more powerful electrostatic interactions between the enzyme and substrate
 Water molecules are sequestered in the active site, allowing an enzyme to eliminate the energy barrier
imposed by the solvent molecules. (Reactions proceed quickly when no solvent molecules interfere)

Not all serine proteases are related by evolution

 The three active sites are identical; proteins diverged from common ancestor and retained overall
structure and catalytic mechanism

Chymotrypsin, trypsin, and elastase have very similar

structures but different substrate specificity
 Specificity due to the specificity pocket: cavity on the
enzyme surface at the active site that accommodates
residue on the N-terminus side of scissile bond

Chymotrypsin is activated by proteolysis

Proteases are limited by the action of protease inhibitors
 Zymogens (inactive precursors) are synthesized by proteases that can be activated later when needed

Chymotrypsinogen: inactive precursor of chymotrypsin

 Synthesized by the pancreas; along with zymogens of trypsin
(trypsinogen), elastase (proelastase)
 They are all activated via proteolysis after secreted into the
small intestine
 Autoactivation: (example) trypsin activation causes activation
of trypsinogen
Reading Assignment: Blood Coagulation requires a cascade of proteases
Loss of blood due to severe trauma can be halted by formation of
clots (aggregated platelets) and a mesh of fibrin protein
 Conversion of fibrinogen into fibrin polymers is the last step in
coagulation – series of proteolytic reactions
 Thrombin (enzyme) cleaves fibrinogen to fibrin, specifically the
peptide bonds following Arg residues
o Circulates as an active precursor (zymogen) called
 Factor Xa catalyzes specific hydrolysis of prothrombin to
generate prothrombin through a series of activated reactions –
Enzyme Cascade
 Coagulation reactions have an amplifying effect – each protease
is a catalyst for the activation of another catalyst.

Protease Inhibitor limit protease activity

Inhibitors pose as protease substrates but are not completely hydrolyzed.

Chapter 7: Enzyme Kinetics and Inhibition

Enzyme Kinetics: fully describe enzyme activity by applying mathematical tools to quantify and enzyme’s
catalytic power and its substrate affinity as well as its response to inhibitors
 The progress of any reaction is expressed as velocity (v).
o The rate of disappearance of the substrate (S) or rate of appearance of product (P)

Enzyme-Substrate (ES) Complex E + S  ES  E + P

 Has a hyperbolic curve that suggests that an

enzyme physically combines with a substrate

 At low substrate concentration, the enzyme

quickly converts all substrate to products
o As more substrate is added, the enzyme
becomes saturated with substrate

 Only for simple enzymes

Rate equations describe chemical processes

Unimolecular Reactions A  B
 Involve only a single reactant
 v = k[A] k = sec-1
 Is a first order reaction: rate depends on concentration of one reactant.
Bimolecular (Second Order Reaction) A + B  C
 Involve two reactions
 v = k[A][B] k = M-1s-1
 Velocity of a second order reaction is proportional to the product of the two reactant concentrations
Michaelis-Menten equation is a rate equation for an enzyme-catalyzed reaction

 Vmax: maximum reaction velocity; when [S] is very high, all the enzyme is in its ES form and approaches
maximum activity
o Vmax = k2[E]T

𝑉𝑚𝑎𝑥 [𝑆]
 Equation: 𝑣0 =
𝐾𝑚 +[𝑆]

 Km is the substrate concentration at which the reaction velocity is half-maximal

o Indicates how efficiently an enzyme selects a substrate to convert to product.
o Measure of an enzyme’s affinity for a substrate
o  Km =  Substrate Affinity

The catalytic constant describes how quickly an enzyme can act

 Catalytic Constant (kcat): rate constant of reaction when the enzyme is saturated with substrate
o Also known as the turnover number: number of catalytic cycles that each active site undergoes
per unit of time
 Number of substrate molecules transformed to product molecules by a single enzyme in
a given period of time.
 Is a first-order rate constant; units = s-1
Kcat/Km indicates catalytic efficienc
 Enzyme effectiveness depends on how avidly in binds substrate and how rapidly it converts them to
 At low concentrates of substrate ([S] < Km),
o Very little Enzyme-Substrate complex (ES) forms and [E] = [E]T
 Kcat/Km (units = M-1s-1; second order reaction)
o The value, more than either Km or Kcat alone, represents the enzyme’s overall ability to convert
subsrate to product.
 An enzyme reaches catalytic perfection when its overall rate is diffusion controlled.
o Diffusion-controlled limit: maximum rate at which two freely diffusing molecules can collide
with each other in aqueous solution
Km and Vmax are experimentally determined
To meet assumptions of the Michaelis-Menten model, the concentration of the substrate must be greater
than the concentration of the enzyme

 Lineweaver-Burk Plot: linear transformation of the velocity versus substrate curve

1 𝐾𝑚 1 1
=( ) +
𝑣0 𝑉𝑚𝑎𝑥 [𝑆] 𝑉𝑚𝑎𝑥

 A plot of 1/v0 versus 1/[S] gives a straight line with

 Slope = Km/Vmax
 Y intercept on the 1/vo axis = 1/Vmax

Not all enzymes fit the Michaelis-Menten Model

1. Multisubstrate Reactions
 Most bisubstrate reactions are redox reactions or transferase reactions

X (oxidized) + Y (reduced)  X (reduced) + Y (oxidized)

 Ex. Transferase reaction catalyzed by transketolase

 Transforms a 6C sugar and 3C sugar to a 4C sugar and 5C sugar.
 Vmax is the max reaction velocity when both substrates are present at concentrations
that saturate their binding sites on the enzyme.
 Random Mechanism: substrates in a bisubstrate reaction that bind in any order but end up in the
active site at the ame time
 Ordered mechanism: enzymes which one substrate must bind before the other
 Ping-pong mechanism: one substrate binds and one product is released before the other substrate
binds and the second product is released.

2. Multistep Reactions
 An enzyme-catalyzed reaction may contain many steps.
 The meaning of Kcat is the same for single-step reactions.
 Ex. Multistep transketolase reaction

3. Nonhyperbolic Reactions
 Oligomeric enzymes with multiple active sites do not obey Michaelis-Menten rate equations
 Allosteric Enzymes: presence of a substrate at one active site can affect the catalytic activity of other
active sites

Cooperative Behavior: occurs when enzyme subunits are structurally inked to each other so that a substrate-
induced conformational change in one subunit causes conformational change.
Allosteric/Regulatory Enzymes: composed of two or more
sub-units that undergo conformational change upon binding of a
 For both catalysis AND regulation
 display sigmoidal curve
 Exhibit cooperativity
 Binding at one active site can affect the catalytic activity of
other site

Enzyme Inhibition
2 classes of inhibitors: irreversible and reversible
Irreversible inhibitor: “Suicide Inhibitor” forms covalent bond or very strong non-covalent interaction with
the enzyme.
 Enter the enzyme’s active site and begins a reaction, but cause an incomplete reaction and get stuck in the
active site.

Reversible Inhibition: results when a substance binds reversibly (noncovalently) to an enzyme to alter its
catalytic properties
 Can easily combine with and dissociate from the enzyme and render it inactive

E+I  EI

 Can effect an enzyme’s Km, Kcat, or both.

 There are 3 common types
I. Competitive
II. Noncompetitive
III. Uncompetitive
Competitive inhibition: the inhibitor directly competes with a substrate for binding to the enzyme’s active


Can happen

 Transition State Analogs: compounds that mimic the transition state to take advantage of the active
site; make better inhibition (competitive inhibition)

Noncompetitive inhibitors: do not compete towards the active site; binds to a site on the enzyme other
than the active site to elicit a conformational change that affects structure/chemical properties of the active
Affect Vmax
Does NOT affect Km
Mixed Inhibition: inhibitor binding to the enzyme alters its conformation in a way that affects both Vmax and

Affects both Vmax and Km

 Less Affinity (Higher Kmax)

Uncompetitive Inhibition: binds to the enzyme-substrate complex; inhibitor can bind after one substrate
has bound to prevent the reaction from continuing and yielding product.

Km decreases as
Vmax decreases
Chapter 11 – Carbohydrates
What are carbohydrates?
Carbohydrates (sugars or saccharides) have the generic formula (CH2O)n : n is greater or equal to 3.
 They are formed from CO2 and H2O.

 Roles include:
o Energy in diet – fuel molecules; glucose and fructose (immediate use), starch and glycogen
(chemical storage forms for future use)
o Mediating intercellular communication – in combination w proteins and lipids on cell surfaces;
informational markers for molecular recognition
 Cell-cell recognition and signal transduction in glycoproteins and glycolipids
o Structural support (cell walls) – scaffolding for bacteria and plant cell wall; connective tissue;
o Components of nucleic acids – structural role as ribose and deoxyribose; and are polar sites for
catalytic processes by RNA ribozymes.

Classification of carbohydrates by length

Monosaccharides – are simple sugars

Small polymers of sugars include:

 Disaccharides: two sugars bound together
 Trisaccharides: three sugars bound together
 Oligosaccharides: “several” sugars bound together

Polysaccharides – large polymers of sugars

Monosaccharides – simple sugars
Glyceraldehyde (Aldose) and dihydroxyacetone (Ketose) are the simplest three carbon sugars.

Monosaccharides can also be classified by the

number of carbon atoms.
 Triose: 3 carbons
 Tetrose: four carbons
 Pentose: five carbons
 Etc.
Most carbohydrates are chiral compounds
Glucose is chiral
glyceraldehyde has exhibited mirror symmetry (2) called enantiomers
Left (L)/Right (D) Enantiomers
D sugars: asymmetric C farthest from carbonyl group (OH on right)
L sugars: chiral carbon farthest from the carbonyl group has (OH on the left)
Epimers - carbohydrates that differ in configuration at one of these

L-amino acids can distinguish D and L prefixes

Cyclization generate 𝛼 and 𝛽 anomers
𝛼 anomer- hydroxyl group lies in opposite side of the ring from the CH2OH group of the chiral carbon that
determines (D) or (L) config.
𝛽 anomer- hydroxyl group points up due to hydroxyl group lies in same side of the ring from the CH2OH group
of the chiral carbon that determines (D) or (L) config.

Not interchangeable
Enantiomers- mirror images of eachother
Epimers- pair of stereoisomers

Anomers in an aqueous solution freely

interconvert between 𝛼 ↔ 𝛽*
*unless hydroxyl group attached to the anomeric carbon is linked to
another molecule

Glucose gets chair conformation and is made of = 64% 𝛽 anomer + 36% 𝛼 anomer

Hexoses and pentoses undergo cyclization DON’T form planar structures

Monosaccharides can be derivatized in many different

Anomeric carbon can undergo oxidation, reducing substances such as Cu(II) →Cu(I)
Benedict’s reagent can distinguish reducing sugar (free monosaccharide)

CH3OH + C6H12O6 (reducing sugar) → Non-reducing sugar

Methyl group can end up 𝛼 or 𝛽 position

Glycosidic bond- links the anomeric carbon to the other group

Glycoside- sugar linked to another molecule

Ribose − − − − 𝑟𝑖𝑏𝑜𝑛𝑢𝑐𝑙𝑒𝑜𝑡𝑖𝑑𝑒 𝑟𝑒𝑑𝑢𝑐𝑡𝑎𝑠𝑒 → 2’ Deoxyribose

One metabolically essential carbohydrate-modifying reaction is the one catalyzed by ribonucleotide reductase
(reduces 2’ –OH group of ribose to convert a ribonucleotide to a deoxyribonucleotide for DNA synthesis)

11-2 Polysaccharides
monosaccharides can are hooked together linked by glycosidic bonds
Only one anomeric carbon involved in the glycosidic bond (C1)
each -OH group can participate in a condensation rxn which permits different bonding arrangements and
allows for branching

Glycans can be sequenced using mass spectrometry

Fault: inability to distinguish isomers
3D structural normally analyzed through NMR
Lactose and sucrose are the most common disaccharide
Disaccharide- 2 monosaccharides linked by glycosidic

● major fuel source for newborn mammals
● broken down by 𝜷 − 𝒈𝒂𝒍𝒂𝒄𝒕𝒐𝒔𝒊𝒅𝒂𝒆 (𝒍𝒂𝒄𝒕𝒂𝒔𝒆)
● adults make little lactase, therefore inefficient in
digestion disaccharide
Galactose + Glucose → Lactose


Glucose + Fructose → Sucrose (𝛼 (1 → 2))

❏ most abundant disaccharide
❏ major form of newly synthesized carbohydrates
Starch and Glycogen are Fuel Storage

𝛼 (1 → 4)
𝛼 (1 → 6)

Amylose: a linear and helical polymer of glucose

Amylopectin: a branched polymer of glucose
Amylopectin 𝜶 (𝟏 → 𝟔)
● plants primary metabolic fuel
● highly branched, easy to add/remove glucose
● compact particle due to 𝛼 𝑙𝑖𝑛𝑘𝑒𝑑 𝑐ℎ𝑎𝑖𝑛𝑠 𝑐𝑢𝑟𝑣𝑒 𝑖𝑛𝑡𝑜 ℎ𝑒𝑙𝑖𝑐𝑒𝑠

Cellulose and Chitin Provide Structural Support

● most abundant polysaccharide
● residues linked 𝛽 (1 → 4)
● extended fibers for rigidity and strength ex. cell
11-1 A Biochemistry Note: Cellulosic Biofuel
wood + agricultural waste → biofuels
● cellulose is never in pure form
● Synthesized by organisms that produce
cellulases (capable of hydrolyzing 𝛽 (1 →
● cellulose normally consist of hemicellulose, pectin, lignin.
biofuel ex. ethanol
approx. 500 - 3,000 residues
● heteropolymer (most abundant being xylose)
● forms a network filled with pectin

● highly hydrophilic due to -OH groups
● holds H2O
● gel-like characteristics

NOT a polysaccharide
● heterogeneous
● aromatic compound lead to difficulty in characterization
● hydrophobic, few -OH groups present
● covalently links to hemicellulose ex. strength in cell walls

All components in wood is a large amount of stored energy into other fuels

● separate the polysaccharide from lignin

● dependent on living organisms or where enzymes were derived from then
● carbohydrate polymers are accessible to hydrolytic enzymes (resulting to mix of monosaccharides)

Glucose + Fungi ( Yeast ) → ethanol

Through fermentation
Bioengineering organism to carry out polysaccharide hydrolysis and convert those monosaccharides to ethanol

● stable
● storable
● transportable
Chitin 𝛽 (1 → 4) 𝑙𝑖𝑛𝑘𝑒𝑑 𝑟𝑒𝑠𝑖𝑑𝑢𝑒𝑠 𝑎𝑟𝑒 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑑𝑒𝑟𝑖𝑣𝑎𝑡𝑖𝑣𝑒𝑠
ex. N-acetylglucosamine

Bacterial Polysaccharides from a biofilm

● do not synthesize cellulosic cell walls
● store fuel as starch or glycogen
● produce extracellular polysaccharides (acts as protective matrix for growth)

Biofilm (an extracellular hydrated polysaccharide)

● attach to surface
● protects from desiccation or from being washed
● harbors community of embedded bacteria
Difficult to characterize biofilm extracellular composition
❖ houses mix of species
❖ proportion of polysaccharide composition depends on many environmental factors

11-3 Glycoproteins
N-linked oligosaccharides
● undergo processing attached to glycoproteins
linked to an Asn side chain
● undergo processing- N-glycosylation begins while a
protein is being synthesized by a ribosome associated
with the rough ER. When the newly synthesized protein
leaves the ER and traverses the Golgi apparatus,
glycosidases and glycosyltransferases act on it

protein is being synthesized by a ribosome associated

with rough ER as translocated into ER lumen
oligosaccharide chain of 14 residues is attached to an Asn
residue (N-linked) leaves ER → Golgi Apparatus →
glycosidases or glycosyltransferases

glycosidases- remove various monosaccharide residues

glycosyltransferases- add new monosaccharide residues

O-linked oligosaccharides
● built in Golgi Apparatus one glycosyltransferases
● tend to be large
● do NOT go undergo processing by glycosidases
● major component of mucus provides protective layer
for respiratory and digestive tracts
● undergo processing attached to glycoproteins linked
to Ser or Thr

Purpose of Oligosaccharide Groups

occupy the protein surface
● hydrophilic
● conformationally flexible
● stabilize protein structure
● intracellular addressing system (deliver newly synthesized proteins to proper cellular location)
● recognition and attachment points (ex. blood type)
The ABO Blood Group System
Best known- ABO Blood Group System
oligosaccharides attached to sphingolipids and proteins on cells
● 15 different carbohydrate configurations on blood group systems

Type A
oligosaccharides has terminal N-acetylated galactose group
● develop antibodies recognize and crosslink red blood cells bearing Type-B oligo.
● can receive Type-O
Type B
oligosaccharides has terminal galactose group
● develop antibodies recognize and crosslink red blood cells bearing Type-A oligo.
● can receive Type-O
Type O
oligosaccharides has none of these terminal groups (mutation- lacking final residue)
● develop antibodies recognize and crosslink red blood cells bearing Type-A or Type-B oligo.
● if A, B, or AB blood fusion is done antibodies will react with transfused cells causing lysis, clumping, or
blocked vessels
● DO NOT develop antibodies recognize and crosslink red blood cells bearing Type-A or Type-B oligo.

Proteoglycans contain long glycosaminoglycan chains

Proteoglycans- protein chain is an attachment site for enormous O-linked polysaccharide called

repeating disaccharides amino sugar N-acetylated and
uronic acid (sugar w/ carboxylate group)

repeating glycosaminoglycans = chondroitin sulfate

● in variably on the extracellular side of the plasma
● attract water (occupy the spaces between the cells
and extracellular matrix ex. collagen fibrils)

Proteoglycans-transmembrane proteins lipid linked

extracellular proteoglycan and glycosaminoglycan chains not connected to protein scaffold play a role in
connective tissue
Mechanical Pressure Squeezes H2O from glycosaminoglycans which allow accommodation to body
● brings negative charged sulfate and carboxylate group of polysaccharide close together
● “shock absorption qualities” spring back repulsion between anionic group is relieved and water drawn
back into the molecule

Bacterial cell-walls- Peptidoglycan

Peptidoglycan- network of crosslinked carbohydrate chains and peptides (make bacteria cell wall)
● gives shape ( surrounds plasma membrane)
● repeating 𝛽 (1 → 4) linked disaccharides
● antibiotics ex. penicillin family block formation of peptide cross-links (killing bacteria)

Staphylococcus aureus
➢ glycans chains are perpendicular to cell surface
➢ honeycomb like
Box 11-C
● inhibits synthesis of DNA, RNA, or proteins
Common role: disruption of cell wall synthesis
Penicillin (𝛽 − 𝑙𝑎𝑐𝑡𝑎𝑚 𝑎𝑛𝑡𝑖𝑏𝑖𝑜𝑡𝑖𝑐)
● first antibiotic used clinically
● inhibits enzyme that cross-link peptidoglycan, this weakening cell wall through disruption of osmotic
MRSA- Bacterias resistant to 𝛽 − 𝑙𝑎𝑐𝑡𝑎𝑚 produce enzyme that cleaves amide bond of 𝛽 − 𝑙𝑎𝑐𝑡𝑎𝑚 𝑟𝑖𝑛𝑔

Chapter 12 – Metabolism and Bioenergetics

ATP – the cell’s energy and is a nucleotide whose reactions have a vital role in metabolism.
Catabolism: break down of molecules to release free energy
 Fats, carbohydrates, and proteins are broken down to CO2, H2O, and NH3
 Production of ATP, NADH, FADH2, and NADPH

Anabolism: building up of molecules by using the free energy released

from catabolism
 Construction of large complex biomolecules from smaller
precursor molecules
Energy is supplied via ATP, NADH, or NADPH

Food and Fuel

Humans are heterotrophs, therefore the food digested and absorbed is a source of metabolic energy and
material for growth and activity. Cells take up products of digestion. The human diet consists of four
biomolecules, where digestion reduces these to smaller molecules:
 Proteins  Amino Acids
 Nucleic Acids  Nucleotides
 Polysaccharides (Carbs)  Monosaccharides
 Fats (Trialglycerols)  Fatty Acids *not technically polymers
Cells take up products of digestion
Digestion is extracellular, in the mouth, stomach and small intestine and catalyzed by hydrolytic enzymes.
 Amylase: hydrolyzes and breaks down starch; consists of linear polymers of amylose and branch polymers
called amylopectin.
 Protease (Trypsin, chymotrypsin, elastase): secreted in the stomach and pancreas which degrade proteins
into small peptides and amino acids.
 Lipase: synthesized in the pancreas and secreted into the small intestine; catalyze the release of fatty acids
from triacylglycerols.

Monomers are stored as polymers

Triacylglycerols (Large globules) are the polymer form of fatty acids in adipocytes. Long-term storage of

Liver and Muscle tissue use monosaccharides to synthesize glycogen (the storage
polymer of glucose)
 Short-term carbohydrates, lasts about 12 hours as a source of energy.
 There is high concentration in liver and muscles.
 Glycogen is highly branched, but compact. Therefore, several glycogen molecules
can clump to form granules.
Branching allows for quick addition or removal of glucose residues.

Biological implication of glycogen branching:

 Each branch in a glycogen molecule has a non-reducing end that allows faster
mobilization of glucose when it is in metabolic demand.
 In conditions of high glucose concentration, it is more efficient to build glycogen due to
the multiple non-reducing ends for expand/building of glycogen for storage.

Amino acids can be used to build peptides

 Because proteins are not dedicated storage molecules for amino acids, excess amino acids cannot be
saved for later.
 Instead, excess amino acids are broken down and are converted into carbohydrates (stored as glycogen)
or converted into acetyl units (Acetyl CoA) for conversion into fats.
 During starvation, proteins are catabolized to supply the body’s energy needs.
 Amino acids and glucose are required for nucleotide synthesis. Asp, Gln, and Gly supply carbon and
nitrogen atoms to build bases.
Fuels are mobilized as needed
Metabolic fuels are molecules that are broken down by processes that make free energy available for cell
activity. These fuels include: amino acids, monosaccharides and fatty acids.
 When fuel molecule supplies are exhausted, the body must mobilize its stored resources by converting its
polysaccharide and triacylglycerol storage molecules (sometimes protein) to their monomeric units.
 Most body tissues, and primarily the CNS, prefer to use glucose as the primary metabolic fuel.
 Therefore, the liver mobilizes glucose by breaking down glycogen.

Glycogen breakdown occurs via phosphorolysis (degradation of glycogen)

 Phosphate breaks the bonds between glucose residues and is catalyzed
by glycogen phosphorylase, releasing residues from the ends of a
glycogen polymer.
 The phosphate from Glucose-1-phosphate is removed before glucose is
released from the liver into circulation.

When glucose runs low, adipose tissue begins to mobilize its fat stores.
Lipase hydrolyzes triacylglycerols in order for fatty acid release into the
 These fatty acids are not water-soluble and can bind to circulating
 The body cannot budget burning fatty acids however, the heart uses fatty
acids as its primary fuel.

Cellular proteins are continuously degraded therefore, there are two major mechanisms for protein
I. Degradation via the Lysosome (an organelle that contains proteases and hydrolytic enzymes)
o Breaks down proteins enclosed in a membranous vesicle.
o Membrane proteins and extracellular proteins are taken up
by endocytosis and are degraded

II. Degradation via Proteasome (barrel-shaped structure where its

inner chamber contains multiple active sites to carry out peptide
bond hydrolysis)
o Proteins can only enter the proteasome after is has been
covalently tagged with ubiquitin.
o Links C-terminus to a lys side chain (at least four ubiquitins)

Metabolic Pathways
Metabolic pathways can be considered as: a series of intermediates or metabolites; a set of enzymes that
catalyze the reactions by which metabolites are interconverted; an energy-producing/requiring
phenomenon; or a dynamic process that can be turned up or down.
Major metabolic pathways share few common intermediates
A handful of metabolites appear as precursors or products in pathways that lead to all other types of

Glycolysis: the pathway that degrades glucose (6-carbon sugar) which is

phosphorylated and split in half, yielding two molecules of glyceraldehyde-3-
phosphate. This is converted into pyruvate (3-carbon molecule), which is
decarboxylated yielding acetyl-CoA (two carbon acetyl groups are linked to
coenzyme A, CoA)
 Glyceraldehyde-3-phosphate, pyruvate, and acetyl-CoA are the key
players in metabolism.
 Fatty acid breakdown yields acetyl-CoA
 Pyruvate can undergo amino-group transfer to yield alanine.

Metabolic pathways include oxidation-reduction reactions.

Catabolism of amino acids, monosaccharides, and fatty acids = oxidation of carbon atoms
Anabolism of amino acids, monosaccharides, and fatty acids = reduction of carbon atoms

Carbon in methane is highly reduced; Carbon in CO2 is highly oxidized.

Fatty acids have Methylene carbons and Carbohydrates (CH2O) have carbons that undergo oxidation.

Oxidation of metabolic fuels causes transfer of electrons to enzyme

cofactors/coenzymes - nicotinamide adenine dinucleotide (NAD+) or
nicotinamide adenine dinucleotide phosphate (NADP+)
 NAD+ = catabolic reactions
 NADP+ = anabolic reactions

Ubiquinone: a lipid-soluble electron carrier in which a membrane-associated enzyme transfers electrons from
a substrate to the electron carrier.
 Ubiquinone can take up one OR two electrons (NAD+ is strictly a
two-electron carrier)
 Reduced ubiquinol can diffuse through membrane and donate its
The citric acid cycle generates considerable amounts of reduced cofactors that are recycled through oxidative
 Reoxidation of NADH and QH2 and production of ATP
requires reduction of O2 and H2O

Basically: NAD+ and ubiquinone collect electrons and free

energy from reduced fuel molecules and are ultimately
transferred to O2; this free energy is in the form of ATP.

Overview of metabolism:
 Monomers are formed.
 Intermediates with two or three carbons are formed.
 Carbons are fully oxidized to CO2.
 Electron carriers gain electrons and are recycled via electron loss.
 ATP and H2O are produced.

(from book)
 Metabolic pathways are all connected
 Pathway activity is regulated
 Not every cell carries out every pathway
 Each cell has a unique metabolic repertoire
 Organisms may be metabolically interdependent.

Humans metabolism depends on vitamins

Vitamins are the building blocks for coenzymes, which
are needed by enzymes to complete reactivity.
 Niacin (Vitamin B3) – precursor for Nicotinamide
Adenine Dinucleotide (NAD+)
 Riboflavin (Vitamin B2) – precursor for FAD (in
oxidation reduction)
 Vitamin C – Precursor for hydroxylation reactions

Fat Soluble Vitamins: A, D, E, K (Cannot have in excess)

Vitamin C: a cofactor for prolyl hydroxylase and lysyl hydroxylase, which are responsible for hydroxylation of
proline and lysine amino acids in collagen.
 Hydroxyproline and hydroxylysine stabilize collagen by cross-linking the propeptides in collagen.
 Deficiency (Scurvy): causes fragile capillary walls, easy bleeding of gums, loosened teeth, and bone-joint
Chapter 13 Glucose Metabolism
Biochemical Roles of Glucose
 Source of metabolic energy
 Precursor for the synthesis of structural polysaccharides (cellulose), disaccharides (sucrose and lactose)
and monosaccharides (galactose and fructose)

All cells of the body utilize glucose for fuel

 Erythrocytes depends solely on glucose
 Brain cells are dependent on a constant supply of glucose.

Glycolysis: ten-step conversion of 6-carbon glucose to a 3-carbon pyruvate.

 Role: universal pathway for extraction of energy from carbohydrates
o Aerobic organisms: initial phase to prepare glucose for additional energy production
o Anaerobic organisms: ATP and NADH is the only significant energy available from carb
 Does NOT require molecular oxygen
 Occurs in the cytoplasm
 Hexose (6C Glucose) is split into 2 molecules of -ketoacid pyruvate (3C)
o Production of a small amount of energy
o Net: 2 ATP, 2 NADH

Glucose + 2 NAD+ + 2 ADP + 2 Pi  2Pyruvate + 2NADH + 2ATP

 Phosphorylation of two molecules of ADP to produce 2 ATP

 Reduction of two molecules of NAD

Steps 1-5: Energy Investment Steps 6-10: Energy Payoff

Energy Investment Phase: Requires the investment of free energy in the form of two ATP molecules

Step 1: Hexokinase Reaction

Hexokinase transfers a phosphoryl group from ATP to the C6 OH group of glucose to form glucose-6-

 Kinase: enzyme that phosphorylates

 Irreversible reaction (Negative standard
free energy change)
o Prevents glucose from
backing out of glycolysis
 ATP is invested; ATP hydrolysis drives
the reaction

Step 2: Phosphoglucose Isomerase Reaction

Isomerization reaction; glucose-6 phosphate is converted into fructose-6-phosphate

 Conversion of a hexose to a pentose

 Reaction is near-equilibrium; freely

Step 3: Phosphofructokinase Reaction

Consumes 2nd ATP molecule via phosphorylation of fructose-6-phosphate to yield fructose 1,6-bisphosphate

 Primary
Control/Regulation point
of glycolysis
 Irreversible Reaction
 Slowest reaction in

Phosphofructokinase response to allosteric effectors

 ADP binds to the enzyme, causing a
conformational change that promotes F-6-P
o ADP concentration in the cell is a good
indicator for the need of ATP.
 Phosphoenolpyruvate (a product of step 9) binds
to PFK, causing a conformation that destabilizes
F-6-P binding, inhibiting catalytic activity.
o PFK acts as a feedback inhibitor to slow the
pathway by decreasing the rate of reaction
when there is plenty of PEP and ATP.
Fructose-2,6-bisphosphate: most potent activator of phosphofructokinase in mammals.
 Synthesized from F-6-P by phosphofructokinase-2
 Phosphofructokinase-2 is hormonally stimulated
when there is high glucose concentration in the
 The increase in F-2,6-bP activates PFK to increase
the flux of glucose in glycolysis.

PFK reaction is rate-determining reaction, which operates far from equilibrium and has a large negative free
change of energy and is irreversible under metabolic conditions.
 Rate of reaction is altered by allosteric effectors, but NOT by fluctuations of concentration of substrates
and products.

Step 4: Aldolase
Converts fructose-1,6-bisphosphate to two three carbon molecules, Dihydroxyacetone phosphate and

 Reverse of an Aldol condensation reaction catalyzed

by aldolase.
 Two catalytically important residues:
 Lys Residue – forms Schiff base (imine) with
 Ionized Tyr residue – acts as a base catalyst

Products of the aldolase reaction are both phosphorylated, but only glyceraldehyde-3-phosphate
proceeds through the pathway

Step 5: Triose Phosphate Isomerase

Dihydroxyacetone phosphate is converted into glyceraldehyde-3-phosphate by triose phosphate isomerase.

 2 molecules of glyceraldehyde-2-phosphate proceed

through glycolysis
 Triose phosphate isomerase is a “catalytically
perfect” enzyme – rate is limited only by the rate at
which its substrates can diffuse to its active site.
 Catalytic mechanism involves low-barrier hydrogen
 Catalytic power of triose phosphate isomerase depends on protein loop that closes over active site
Energy payoff phase: 4 ATP molecules are produced; net gain of 2 ATP; involve three-carbon
Step 6: Glyceraldehyde-3-phosphate Dehydrogenase
Glyceraldehyde-3-phosphate is both
oxidized and phosphorylated into 1,3-

 Addition of inorganic phosphate to

the substrate (rather than
phosphorylation via ATP) – group
transfer reaction.

 Oxidation-Reduction reaction
o NAD+ is reduced to NADH
o Aldehyde group of GA-3-P is oxidized

Glyceraldehyde-3-phosphate dehydrogenase catalyzes the removal of an H-


This reaction is inhibited by arsenate (AsO43-), which competes with Pi (PO43-)

for the Cys residue in the active-site.

Step 7: Phosphoglycerate Kinase

1,3-Bisphosphoglycerate donates a phosphoryl group to ADP to produce 3-phosphoglycerate and ATP
catalyzed by phosphoglycerate kinase.

 Reaction occurs twice: 2 ATP have been

 Kinase transfers phosphoryl group
 Substrate-Level Phosphorylation

Step 8: Phosphoglycerate mutase

3-phosphoglycerate is converted to 2-phosphoglycerate.

 Isomerization of 3-PG to 2-PH requires an enzyme

active site that contains a phosphorylated His residue.
o The phosphor-His transfers phosphoryl group
to 3-phosphoglycerate to generate 2,3-
bisphosphoglycerate, which gives a phosphoryl
group back to the enzyme leaving 2-
phosphoglycerate and phospho-His
Step 9: Enolase
2-Phosphoglycerate is converted to phosphoenolpyruvate and H2O via dehydration reaction

 Enzyme active site includes an Mg2+ ion that

coordinates with the OH group at C3, making a
better leaving group.

 Fluoride ion and Pi forms a complex with Mg2+,

which can inhibit the enzyme.

Step 10: Pyruvate Kinase

Converts phosphoenolpyruvate to pyruvate and transfers a phosphoryl group to ADP to produce ATP.

Reaction occurs in two parts:

1. ADP attacks phosphoryl group of PEP to form ATP and enolpyruvate
2. Tautomerization of enolpyruvate to pyruvate; highly exergonic

Substrate-Level Phosphorylation

Substrate-level phosphorylation vs. Oxidative Phosphorylation

 Substrate-level: process of forming ATP by phosphoryl transfer from a reactive intermediate
 Oxidative: process for making ATP from ADP by electron transfer linked to respiration.

Regulatory enzymes of Glycolysis

I. Hexokinase: catalyzes irreversible reaction and subject to inhibition by glucose-6-phosphate (product)
II. Phosphofructokinase
a. Inhibition via phosphoenolpyruvate and activation via ADP – Bacteria
b. Activation via Fructose-2,6-bisphosphate
III. Pyruvate Kinase
 Feed-forward Activation
 Fructose-1,6-bisphosphate activates pyruvate kinase at an allosteric site.
Pyruvate Metabolism
Pyruvate can be further broken down to either:
 Acetyl-CoA
 Used to synthesize compounds such as oxaloacetate

Pyruvate can metabolize depending on the organism, type of cell, and intracellular conditions of the cell.
 In oxygen-plentiful conditions: pyruvate is completely oxidized to CO2 and electrons are ultimately
transferred to O2
 In oxygen-limited conditions: other molecules serve as electron acceptors to regenerate NAD +

Fermentation: extraction of energy from carbohydrates and other organic substrates without using oxygen as
an electron acceptor
a. Lactate Fermentation: anaerobic microorganism and
in muscle cells under limiting oxygen conditions
b. Ethanol fermentation: yeast and other

Yeast Fermentation
 Sugars are transformed to pyruvate via glycolysis
 Then, pyruvate decarboxylase removes carboxylate group
on pyruvate to produce acetaldehyde
 Alcohol dehydrogenase then reduces acetaldehyde to ethanol.

Alcohol metabolism
The liver can metabolize ethanol, which is readily absorbed in the GI tract and transported by the blood
 Alcohol dehydrogenase converts ethanol to acetaldehyde
 Then, acetaldehyde is converted to acetate via acetaldehyde hydrogenase.
 Both require NAD+ as a cofactors.

Ethanol is considered mildly toxic.

 Toxicity of acetaldehyde and acetate in liver and brain tissue.
 Ethanol can induce vasodilation – flushing; warming and reddening of skin due to increased blood flow
 Heart and respiration rate is decreased
 Kidneys increase excretion of water – ethanol interferes with ability of hypothalamus to sense osmotic
 Stimulation of signaling from neurotransmitters that function as ligand-gated ion channels – inhibits
neuronal signaling.

Hangover is a result of production of acetaldehyde and acetate.

 Production results in NAD+ consumption – lowers NAD+:NADH ratio
 Insufficient NAD+  diminished ATP production via glycolysis (NAD+ required for glyceraldehyde-3-
phosphate dehydrogenase)

Shortage of liver NAD+ slows fatty acid breakdown and promotes fatty acid synthesis
Further catabolism of pyruvate is initiated via decarboxylation
to form a two-carbon acetyl group linked with coenzyme A
 Acetyl-CoA is a substrate for the citric acid cycle.

Pyruvate is a precursor for several molecules.

 The two-carbon unit Acetyl-CoA (derived from pyruvate) can be used for fatty acid synthesis.

 Pyruvate is also a precursor for oxaloacetate – an intermediate in

Amino Acid synthesis, gluconeogenesis, and the TCA cycle
o Oxaloacetate is synthesized by pyruvate carboxylase
o Pyruvate carboxylase has a biotin prosthetic group
(carries CO2) – covalently linked to a Lys residue

The liver can synthesize glucose from non-carbohydrate precursors via gluconeogenesis.
 Kidneys can perform gluconeogenesis (limited) when glycogen supply in liver is exhausted.

It is the reverse of glycolysis: conversion of two molecules of pyruvate to one molecule of glucose.
 Gluconeogenesis bypasses pyruvate kinase, phosphofructokinase, and hexokinase.
 Instead four enzymes are used in this pathway:
o Hexokinase (in glycolysis)  Glucose-6-Phosphatase (in gluconeogenesis)
o Phosphofructokinase (in glycolysis)  Fructose bisphosphatase
o Pyruvate kinase (in glycolysis)  Phosphoenolpyruvate carboxykinase & Pyruvate carboxylase
Pyruvate cannot be directly converted to phosphoenolpyruvate (pyruvate kinase catalyzes irreversible
reaction – step 10 in glycolysis)
1. Pyruvate is first carboxylated by pyruvate
carboxylase to yield oxaloacetate (this utilizes ATP.
2. Oxaloacetate is then decarboxylated via
phosphoenolpyruvate carboxykinase to form

Pyruvate carboxylase consumes ATP; phosphoenolpyruvate carboxykinase consumes GTP

 Cleavage of phosphoanhydride bonds is required to supply free energy to convert.

Alanine and Glutamine) are the main sources of precursors for gluconeogenesis because they can be
converted to oxaloacetate and then to phosphoenolpyruvate.
 During starvation, proteins are broken down and are used to produce glucose to fuel the CNS.

Two molecules of phosphoenolpyruvate are converted to one molecule of fructose-1,6-bisphosphate in a

series of six reversible reactions.

The final three reactions of gluconeogenesis require two enzymes.

1. First step undoes phosphofructokinase – fructose bisphosphatase hydrolyzes C1 phosphate of fructose-
1,6-bisphosphate to yield fructose-6-phosphate
2. Phosphoglucose isomerase produces glucose-6-phosphate (reverse of step 2)
3. Glucose- 6-phosphatase catalyzes hydrolytic reaction that yields glucose and inorganic phosphate (Pi)

Regulation of gluconeogenesis – Fructose Bisphophosphatase Step

Simultaneous occurrence of glycolysis and gluconeogenesis is energy wasting – there is regulation of opposing
 Combination of these metabolic reactions is a futile cycle (have no useful result) – provides a mean of fine-
tuning the output of a metabolic pathway.
 Major regulatory point: interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate

When concentration of fructose-2,6-bisphoshphate is high:

 Glycolysis is stimulated
 Gluconeogenesis is inhibited.

Fructose-2,6-bisphosphatase modulates activity of both fructokinase and fructose bisphosphatase – as activity

of one enzyme increases, the other decreases – dual regulatory effect.
Glycogen Synthesis and Degradation
Glucose produced by gluconeogenesis is stored in the liver and other tissues as glycogen; glucose units can be
removed from glycogen via phosphorolysis.
 Glycogen degradation is thermodynamically spontaneous.
 Glycogen synthesis requires input of free energy.

Glycogen synthesis consumes free energy of UTP

Glycogen is composed of monomers of glucose-1-phosphate, which
is isomerized by phosphoglucomutase

Glucose-1-phosphate is activated by the reaction of UTP to form UDP-glucose

 This is a reversible phosphoanhydride exchange.

Glycogen synthase: transfers a glucose unit to the C4-OH group at

the end of a glycogen branch – extends the linear (14)-linked

Glycogen is phosphorolyzed to yield glucose-1-phosphate.
In liver – phosphoglucomutase converts glucose-1-phosphate to glucose-6-phosphate, then glucose-6-
phosphatase hydrolyzes the release of free glucose from glucose-6-phosphate

 Linear chains are broken down via phosphorolysis.

 Branched chains are broken down via hydrolysis
 Glucose-6- phosphate can enter glycolysis at step 2 – one less ATP is consumed = higher net ATP
Pentose Phosphate Pathway (PPP): converts glucose-6-phosphate to ribose-5-phosphate
 Is an oxidative pathway
 Generates NADPH

*Starting point = glucose-6-phosphate

1. Glucose-6-phosphate dehydrogenase catalyzes irreversible transfer

of hydride ion from glucose-6-phosphate to NADP+ to form a 6-
phosphoglucono-lactone and NADPH

 Deficiency in G6P-D causes decreased production of NADPH,

interfering with normal oxidation-reduction processes – makes cells
more susceptible to oxidative damage
o More resistant to malaria

2. 6-phosphogluconolactonase hydrolyzes the lactone intermediate to


3. 6-phosphogluconate dehydrogenase oxidatively decarboxylates 6-

phosphogluconate to ribulose-5-phosphate
 Two molecules of NADP are produced

4. Ribulose-5-phosphate isomerase isomerizes ribulose-5-phosphate

to ribose-5-phosphate
 Ribose-5-phosphate is a precursor for the ribose unit in nucleotides

The PPP produces ribose and NADPH (reducing agent)

for the reduction of ribose to deoxyribose via
ribonucleotide reductase.

The need for NADPH can be greater in other reactions – excess carbons are recycled into intermediates for
glycolysis for degradation into pyruvate or for use in gluconeogenesis.
 Enzymes such as transketolase and transaldolase – transfer 2/3C units among intermediates to produce
three to seven carbon sugars.
 Ex. Transform 5C-Ribulose into 6C-units (fructose-6-phosphate) and 3C-unites (glyceraldehyde-3-
Chapter 14 – The Citric Acid Cycle
Beri-Beri Disease: Nutritional disease caused by the deficiency of thiamine (Vitamin B1)
 The activated form of B1 is thiamine pyrophosphate (TPP), which is a coenzyme that assists enzymes
involved in decarboxylation.

Vitamin B1 (Thiamine) is a precursor for thiamine pyrophosphate, a cofactor used by the pyruvate
dehydrogenase complex (PDH)
 It is also a co-factor for -ketoglutarate dehydrogenase
 And is also found in a variety of foods such as meat and grains.
 Decarboxylation reactions occur as pyruvate is further oxidized in the TCA,
therefore TPP-B1/Thiamine is a required cofactor for decarboxylase.

3C pyruvate to a 2C acetyl with loss of CO2

 2C acetyl + 4C Oxaloacetate = 6C
 6C to 5C with loss of CO2
 5C to 4C with loss of CO2

Preliminary Phase

Bridge between glycolysis

and aerobic metabolism

Central and Universal pathway

of aerobic metabolism

The citric acid is central metabolic pathway whose starting material is 2C-acetyl units derived from amino-
acids, monosaccharides, and fatty acids. These are oxidized to waste product CO2, with the reduction of
cofactors: NAD+ and ubiquinone (Q)
 Pyruvate is the starting point of the citric acid cycle (end product of glycolysis)

Mitrochondria: the powerhouse of the cell

 Has an inner matrix, where the electron transport chain is located
 Has an intermembrane space
 The citric acid cycle takes place in the mitochondria of eukaryotes
The Pyruvate Dehydrogenase Complex Reaction
: A multi-enzyme complex used to make chemical transformations economical, efficient, rapid, and prevents
unwanted side reactions – catalyzes the decarboxylation of pyruvate, which is oxidized into 3 CO2 (1st CO2 is
released when pyruvate is decarboxylated to an acetyl unit; the 2nd and 3rd are products of the citric acid cycle)
 The PDH complex is a metabolon
 It is located in the mitochondria
Pyruvate translocase: is a permease that functions by exchanging pyruvate and hydroxide ions, in order to
balance the electrical charge on each side of the membrane

The pyruvate dehydrogenase complex directs conversion of pyruvate to acetyl CoA – this is the bridge
between glycolysis and aerobic metabolism
 Pyruvate (3C)  Acetyl CoA (2C) + NADH + CO2 Cytosol Mitochondria

The oxidation of pyruvate requires 3 enzymes (E1, E2, NAD+ NADH + H+

E3), 5 coenymes, and 5 distinct reactions.
The chemical transformation of pyruvate involves:
 Decarboxylation (Loss of CO2) PDC
 Oxidation of the keto-group of C2 to a carboxyl Acetyl CoA
group Pyruvate CO2 CoenzymeA
 Activation by linkage through a thioester bond to
coenzyme A

The Pyruvate Dehydrogenase Complex happen in 5 Steps (*don’t have to

know, FYI only)
1. E1 decarboxylates pyruvate; CO2 is released.
2. A leftover hydroxyethyl group is transferred to E2.
3. An acetyl group is transferred to CoA.
4. E2 is restored.
5. E3 is restored and NADH is produced.

Coenzyme A (CoA) has Vitamin B5 – they help the body convert food (carbohydrates) into fuel (glucose),
which is used to produce energy. These B vitamins are referred to as B complex vitamins, which also help the
body use fats and proteins.
 The energy stored in the
thioester bond drives the
unfavorable reactions.

 Use of CoASH has an acetyl (TCA) and acyl

carrier (fatty acid metabolism)
o Fatty Acids + CoASH  Fatty AcylCoA
Dehydrogenases is Metabolism via different Co-Enzymes
 If alcohol to aldehyde  Alcohol Dehydrogenase  NADH + Aldehyde
 If single bond to DB bond  Dehydrogenase  DB bond + FADH2

The Citric Acid Cycle (Eight Reactions)

The citric acid cycle is a central and universal pathway of aerobic metabolism
 Also known as the TCA and Krebs cycle
 Occurs in the mitochondrial matrix
 It is considered amphibolic – it is both catabolic and anabolic
 Degradation of acetyl-CoA (from pyruvate and other sources) to CO2
 Supplying precursors for biosynthesis of amino acids, porphyrins, and purine and pyrimidine bases for
 Production of energy-rich molecules: GTP, NADH, and QH2

Acetyl-CoA is derived from pyruvate and is a product of amino
acid catabolism (carbon skeletons of amino acids are broken
down to pyruvate)
Acetyl-CoA is a direct product of degradation of certain amino
acids and of fatty acids

Acetyl Co-A enters the TCA cycle for further oxidation.

 Process is highly exergonic and free energy is conserved via
GTP and reduced cofactors.

For each acetyl group, two molecules of fully oxidized CO2 are
produced – loss of 4 pairs of electrons.
 These electrons are transferred to 3NAD+ and ubiquinone
to produce 3 NADH and 1 QH2.

Acetyl-CoA + GDP + Pi + 3 NAD+ + Q  2 CO2 + CoA + GTP + 3 NADH + QH2

The TCA is a high-energy-generating cycle

 1 NADH = 2.5 ATP
 1 QH2 = 1.5 ATP
Reaction 1: Citrate Synthase
Adds an acetyl group to oxaloacetate – the acetyl group of Acetyl-CoA
condenses with 4C-Oxaloacetate to produce Citrate

Citrate synthase is a dimer that undergoes conformational change upon

binding of oxaloacetate, which allows the binding of Acetyl-CoA
 Can synthesize a C-C bond without a metal ion cofactor
 The first reaction intermediate is stabilized by formation of low-barrier hydrogen bonds
 The coenzyme A released can be reused by the PDH or be used in the TCA to synthesize succinyl-CoA.

The free energy of the reaction is highly exergonic, citrate synthase has a free energy equivalent to breaking
the thioester bond of Acetyl-CoA

Reaction 2: Aconitase
Catalyzes the reversible isomerization of citrate to isocitrate
 The reaction intermediate is Aconitate.

Citrate is a symmetrical molecule – but only 2 carboxymethyl

arms undergo dehydration and rehydration, they are no longer
identical when bound to an asymmetrical enzyme

Reaction 3: Isocitrate dehydrogenase

Releases the first CO2 – is the oxidative carboxylation of
isocitrate to -ketoglutarate
 The substrate is oxidized accompanied by the reduction of
 The carboxylate group that is  to the ketone function (meaning that is two carbons away from the
ketone) is eliminated as CO2
o An Mn2+ ion in the active site helps stabilize the negative charges of the intermediates.
 The CO2 molecules generated (along with CO2 in the next reaction and CO2 produced by decarboxylation of
pyruvate) are diffused out of the cell and carried in the bloodstream to the lungs.
o Carbons are oxidized; NAD+ is reduced.

Energy released from the cleavage of the thioester


There is a
push via a
3 alcohol
Reaction 4: -ketoglutarate dehydrogenase
Releases the 2nd CO2 – catalyzes an oxidative decarboxylation reaction,
transferring the remaining 4-carbon fragment to CoA
 -ketoglutarate dehydrogenase is a multienzyme complex that
resembles the PDH complex in structure/mechanism; E3 is the same
enzyme in both complexes

Free energy of oxidation -

ketoglutarate is conserved in
formation of the thioester

Fates of carbons in the Citric Acid Cycle

 Two carbon atoms are lost as CO2 – catalyzed by isocitrate dehydrogenase (step 3) and -ketoglutarate
dehydrogenase (step 4) are not the same carbons that entered the cycle as Acetyl-CoA.
 The acetyl carbons become part of oxaloacetate and are lost in the reactions

Reaction 5: Succinyl-CoA synthetase

Also called succinate thiokinase Succinyl-CoA
 Catalyzes substrate-level phosphorylation
 Cleaves the high-energy thioester bond of succinyl CoA
 Reaction is couple to phosphorylation of GDP to GTP

Reaction 6: Succinate Dehydrogenase

Succinate is oxidized to fumarate – reversible dehydrogenation
 The oxidation-reduction reaction requires an FAD prosthetic
group, which is reduced to FADH2

Regeneration of the enzyme requires re-oxidation of FADH2.

 Enzyme is embedded into the inner mitochondrial membrane,
therefore is reoxidized by the lipid-soluble electron carrier, ubiquinone (Q) – acquires 2 electrons to be
QH2 (ubquinol)
Reaction 7: Fumarase
(Also fumarate hydratase)
Catalyzes the reversible hydration of a double bond
to convert fumarate to malate

Reaction 8: Malate Dehydrogenase

The last reaction is the regeneration of oxaloacetate from malate via an
NAD+-dependent oxidation reaction

Energy-generation of the Citric Acid Cycle

The entire pathway catalytically disposes carbon atoms derived from amino acids, carbohydrates, and fatty
 Each NADH yields approximately 2.5 ATP
 Each QH2 (of FADH) yields approximately 1.5 ATP
 Every acetyl unit that enters the TCA cycle can generate a total of 10 ATP equivalents.
A muscle operating anaerobically produces only 2 ATP per glucose; a muscle operating aerobically produces 32
ATP equivalents per glucose – Pasteur effect (rate of glucose consumption by yeast decreased when cells were
shifted from aerobic to anaerobic conditions)
Summary of The Citric Acid Cycle
1. Acetate (C2) enters as Acetyl-CoA and 2 carbons
leave the cycle as CO2
2. 3 NAD+ are reduced to NADH by dehydrogenase
catalyzed reactions
3. 1 FAD is reduced to FADH2

Regulation of the Citric Acid Cycle at three irreversible

The flux through the TCA is regulated at three metabolically irreversible steps
1. Citrate synthase reaction (Reaction 1)
2. Isocitrate dehydrogenase reaction (Reaction 3)
3. -ketoglutarate dehydrogenase (Reaction 4)
Citrate synthase
 Inhibited by citrate
 Citrate also inhibits PFK (in glycolysis) which decreases the
supply of Acetyl-CoA
 Inhibited by NADH

Isocitrate dehydrogenase
 Inhibited by NADH
 Activated by ADP and Ca2+

-ketoglutarate dehydrogenase
 Inhibited by NADH and Succinyl-CoA
 Activated by Ca2+

The intermediates produced by the citric acid cycle are biosynthetic

precursors for other molecules.
Ex. -ketoglutarate can undergo reductive amination by glutamate
dehydrogenase to produce glutamate
 Glutamate is also the precursor for glutamine, arginine and proline.
 Any of the TCA intermediates can be converted to oxaloacetate, which can serve as gluconeogenic
Anaplerotic reactions replenish TCA intermediates
Pyruvate + CO2 + ATP + H2O  Oxaloacetate + ADP + Pi

Citrate and pyruvate can cross the mitochondrial membrane via specific
transport proteins
 This allows carbon atoms from mitochondrial Acetyl-CoA to be
transferred to the cytosol for fatty-acid/cholesterol synthesis

Intermediates diverted to other pathways can be

 Oxaloacetate can be replenished by pyruvate and
amino acids
 -ketoglutarate can be replenished by amino
 Succinyl-CoA can be replenished by amino acids
and odd-chain fatty acids.

o Acetyl-CoA activates pyruvate carboxylase so that more oxaloacetate is produced when there is low
TCA activity and accumulation of Acetyl-CoA
 There is normally low oxaloacetate concentration because the malate dehydrogenase reaction
is thermodynamically unfavorable
o The replenished oxaloacetate is converted to citrate, isocitrate, -ketoglutarate, etc.

The TCA cycle is a catalyst, therefore increasing concentrations of its components increases the flux through
the pathway.

Degradation of odd-numbered fatty-acids yields succinyl-CoA

 Degradation of amino acids can also produce some amino acids, -ketoglutarate, succinyl-CoA, fumarate
and oxaloacetate – transaminations

During vigorous exercise, the concentration of TCA intermediates increases 3 to 4-fold in a few minutes to
help increase the energy-generating activity of the TCA cycle.
 There is also an increased activity of the TCA regulatory enzymes (citrate synthase, isocitrate
dehydrogenase, and -ketoglutarate dehydrogenase) which increases the flux 100-fold

 Some glutamate can be converted to -ketoglutarate to boost activity of the TCA cycle
Chapter 15 – Oxidative Phosphorylation
Oxidation of reduced compounds (NADH, FADH2/QH2) in the ETC coupled to ATP
 Oxidation is spontaneous and energy released is conserved in a proton gradient.
 Protons are pumped from the matrix to the intermembrane space.

Oxidative phosphorylation is a more indirect process in which free energy is

converted to (or conserved as) a transmembrane gradient of proons that is then
used to drive ATP syntehses.

Electrons removed from nutrients

are transferred by dehydrogenases
to the cofactors, NAD+ and FAD.

Reduction potential indicates a substances tendency to accept electrons.

 Standard Reduction potential (’): affinity of a substance (such as Ubiq.) for
 The greater the value of ’, the greater the tendency of the oxidized form of
the substance to accept electrons and become reduced

Electrons flow spontaneously from substances with low to high reduction potential (from negative to a more
positive reduction potential)
 Ex. The ’ for NAD+ (-.315V) is lower than in ubiquinone (0.045V), therefore NADH will tend to transfer its
electrons to ubiquinone (NADH is oxidized, Q is reduced)
 Increase reduction potential = increase
electron affinity
 The larger the difference in reduction
potential the change in free energy = more
Mitochondrial Electron Transport
Electrons are shuttle from NADH to O2 in a multistep process that offers several opportunities to conserve the
free energy of oxidation
 Outer membrane- very porous, permits the transmembrane diffusion of substances with masses up to
about 10 Kd
 Composition of the matrix differs from the intermembrane space
Ionic composition of the intermembrane space is equivalent to the cytosol due to the
presence of the porins in the outer membrane
 The relatively impermeable inner mitochondrial membrane encloses the protein-
rich matrix – prevents the transmembrane movements of ions and small
 Individual mitochondria can move around the cell and undergo fusion and fission

Transport of cytosolic NADH – Malate Aspartate and Glycerol-3-Phosphate Shuttle

Much of the cell’s NADH and QH2 is generated by the TCA and fatty acid oxidation, which transfer electrons to
the protein complexes of the respiratory ETC, such as Q, Complex I, Complex II, and Cyt C.
 However, the NADH produced by glycolysis
and other oxidative processes in the cytosol
cannot directly reach the mitochondria
(respiratory chain)
 There is not a transport protein present to
ferry NADH across the inner mitochondrial
membrane (where ETC takes place) – instead
“Reducing equivalents” are imported into the
matrix: Malate-Asparatate Shuttle

Mitochondria also need a mechanism to export ATP and to import ADP and Pi
 Adenine nucleotide translocase exports a ATP and imports ADP, binding one or the other and changing its
conformation to release the bound nucleotide on the other side of the membrane
 Pi is imported from the cytosol in symport with H+
 The protein complexes that carry out ETC and ATP synthesis are oriented in the inner mitochondrial
membrane so that they can bind the NADH

ATP translocase protein Symport protein permits

imports ADP, exports ATP simultaneous movement
of Pi and H+
Complex I transfers electrons from NADH to Q
Complex I is the largest of the ETC (NADH Dehydrogenase)
 45 different subunits, L-shaped protein with numerous
transmembrane helices and a peripheral arm
 ETC takes place in the peripheral arm, which includes
several prosthetic groups that undergo reduction as they
receive electron to become oxidized as they give up their
electrons to the next group

Redox centers- prosthetic groups that undergo reduction and

oxidation within the complex (E* between NAD and Q)
 Allows to form a chain where electrons travel a path of increasing reduction potential

Two electrons donated by NADH are picked up by Flavin mononucleotide (FMN)

 FMN then transfers the electrons one at a time to Fe-S complex (9 total)
 Electrons travel between several Fe-S clusters (1 e carrier) before reaching ubiquinone
 Ubiquinone is a two-electron carrier, but it accepts one electron at a time from an Fe-S donor

As electrons are transferred from NADH to Q, Complex I transfers 4 H+ from the matrix to the intermembrane
 Each proton passes from one side of the membrane to the other via a proton wire
 Proton wire - series of hydrogen-bonded protein groups plus water molecules that form a chain through
which a proton can be relayed
Proton taken up from the matrix are not the same ones that are released into the intermembrane space

Complex II – Other oxidation reactions

contribute to the QH2 pool
The reduced quinone product of Complex I joins a pool of
reduced quinones which are augmented by the activity of
other Redox reactions

Complex II (Succinate dehydrogenase)

 Embedded in the inner membrane – not soluble in the
mitochondrial matrix
 Produces QH2 in the TCA cycle
 Complex II is more like a tributary – does not
undertake proton translocation and therefore does
not directly contribute the free energy of redox reaction towards ATP synthesis (a)
Another source of QH2 is fatty acid oxidation via the oxidation of a C-C bond in a fatty acid catalyzed by acyl-
CoA dehydrogenase (b)
Electrons from cytosolic NADH can also enter the Q-pool through cytosolic/mitochondrial glycerol-3-
phosphate dehydrogenase – shuttles electrons from NADH to Q (c)
 Bypasses complex I
FADH2 goes from C2 to Q, only makes 1.5 ATP because only 6 H+ are pumped into the intermembrane space
Cystolic NADH can start from C1 (heat and liver), which yields 2.5 ATP because 10 H+ are pumped into the
intermembrane space

Cystolic NADH can also go straight to Q (skeletal and brain), where it would only yield 1.5 ATP because 6 H+
are pumped into the inner membrane space (mitochondrial G-3-P dehydrogenase)
G-3-P dehydrogenase is embedded in the inner membrane
C1 = 4 H+, C3 = 4 H+, C4 = 2 H+

Complex III (Q:cytochrome c oxidoreductase/cytochrome bc1)

Ubiquinol (QH2) is reoxidized by Complex III

 Transfers electrons to the peripheral membrane
protein cytochrome C
 Complex III contains 2 cytochromes (b and c1)
 Functional Core: cytochrome b, c1, and iron-sulfur
protein (Rieske protein)
Cytochrome c transfers one electron at a time from C3 to C4

Flow of electrons through Complex III is complicated – two electrons donated by ubiquinol must split up to
travel through a series of one-electron carriers that include cyt c1 and cyt b
 C3 has two active sites where Quinone cofactors undergo reduction and oxidation
Q cycle Net Result
 Two electrons from QH2 reduce two molecules of cytochrome C
 Four protons are translocated to the intermembrane space: 2 from QH2 in the first round of the Q cycle and
two from QH2 in the second round

Complex IV (Cytochrome c oxidase)

Cytochrome c ferries electrons between Complex III & IV
 Is soluble in the intermembrane space
Complex IV is the last enzyme to deal with the electrons
derived from the oxidation of metabolic fuels
 Four electrons delivered by cytochrome C are
consumed in the reduction of molecular oxygen to
 Relays four additional protons from the matrix to the
IM space – two protons for every pair of electrons
The production of water and the proton relays both deplete the matrix proton concentration and contribute
to the formation of a proton gradient across the inner mitochondrial membrane.
Free Radicals and aging
Partial reduction of O2 by Complex IV or the other complexes can produce superoxide free radical O2-
 Can chemically alter nearby molecules – most of the damage is felt by the mitochondria, its lipids, proteins
and DNA are susceptible to oxidation as superoxide steals and electron.
As damage accumulates, mitochondria become less efficient to go through apoptosis
 Oxidative damage mediated by O2- and other free radicals is responsible for the degeneration of tissues
that occurs with aging and is associated with Parkinson’s Disease
and Alzheimer’s Disease
 All cells are equipped with antioxidant mechanisms
Superoxide dismutase converts a superoxide to a less toxic product,
Ascorbate and alpha-tocopherol may protect cells form oxidative
damage by scavenging free radicals
 People that exercise regularly do not exhibit oxidative damage
 Weekend warriors are more susceptible to oxidative damages


E from the H+ concentration gradient is used to make ATP via ATP synthase
 Energy is conserved in proton gradient during ETC
 Intermembrane space has a higher H+ concentration (lower pH)
 Matrix has a lower H+ concentration (higher pH)
 For a gradient to be created, ETC required
 For ATP synthase to function, need H+ gradient
 Proton wires: where H+ travel from matrix to intermembrane space (endergonic) overall spontaneous
Chemiosmotic Theory: proton translocating activity of ETC complexes in the inner mitochondrial membrane
generates a proton gradient across the membrane
 Protons cannot diffuse back into the matrix because the inner membrane is impermeable to ions
 Proton motive force- the imbalance of protons represents a source of free energy; this can drive the
activity of ATP synthase
The imbalance of protons, a nonequilibrium state, has an associated free E the force that would restore the
system to equilibrium)
 Proton gradient has 2 components: reflecting the difference in the concentration of the chemical species
and the difference in electrical charge of the positively charged protons = electrochemical gradient
 Matrix is more negative and intermembrane is more positive
 10 protons translocated from the matrix to the intermembrane space is 200 kJ; therefore, going back it
would be -200 kJ, which is enough to drive the phosphorylation of several molecules of ADP
ATP synthase

F1 (matrix) component has 3 alpha and 3 beta subunits surrounding a central shaft; only the beta serves
catalytic role; catalytic subunit
 F0 (integrated in membrane) includes and A and two b subunits that extent upward to interact with the F1
component and a ring of c subunits; channel for H+
 Mitochondrial ATP synthase has 8 c subunits
 Proton transport through ATP synthase involves the rotation of the c ring pass the stationary a subunit
 3H+ needed to make one ATP, full rotation makes 3 ATP
 A c subunit can take a proton from the intermembrane space
 A slight rotation of the c ring brings another c subunit into position so that it can release its bound proton
into the matrix
 All six subunits of F1 can bind adenine nucleotides, only beta subunits have catalytic activity
 The alpha/beta hexamer change their conformations as the y subunit rotates (like a shaft driven by the c
ring “rotor”)
The binding change mechanism explains how ATP is made

 ATP synthase uses mechanical energy to form a chemical bond (converts mechanical
to chemical energy)
 The interaction between the y subunit and the alpha/beta hexamer explains this
energy transduction
 Rotation-induced conformational change drives unfavorable ATP synthesis reaction
 Binding change mechanism- rotation-driven conformational changes alter the affinity
of each catalytic beta subunit for an adenine nucleotide
 At any moment, each catalytic site has a different conformation (and binding affinity),
referred to as the open, loose, or tight state
1. The substrates ADP and Pi bind to a beta subunit in the loose state
2. The substrates are converted to ATP as rotation of the y subunit causes the beta subunit to shift to the
tight conformation
3. The product ATP is released after the next rotation, when the beta subunit shifts to the open
 Because the three beta subunits of ATP synthase act cooperatively, they all change their
conformation simultaneously as the y subunit turns
 A full rotation of 360 degrees is required to restore the
enzyme to its initial state, but each rotation of 120 degrees
results in the release of ATP from one of the three active
 In the absence of a proton gradient, no ATP is synthesized
because there is no free E to drive the rotation of the y

P:O ration describes the stoichiometry of oxidative


 Chemical energy is transduced to a proton motive force, then

to the mechanical movement of a rotary engine, and finally
back to chemical energy in the form of ATP
 In oxidative phosphorylation the regulated set would be the
reaction catalyzed by complex 4 (cytochrome c oxidase)
 Oxidative phosphorylation is regulated primarily by the
availability of reduced co-factors NADH and FADH2 (QH2)
Ch. 8 Lipids
Lipids: are defined primarily by the absence of functional groups
 Non-polymeric
 Lack the ability to form hydrogen bonds
 Bulk of the structure is hydrocarbon like
 Several classifications:
o Fatty Acids
o Triacylglycerols
o Glycerophospholipids
o Sphingolipids
o Isoprenoids: Cholesterol and Terpenes

Functions of lipids:
1. Energy Metabolism: long term storage energy - non-polar lipids
2. Components of biological membranes – polar lipids with nitrogen
and phosphate
3. Precursor of hormones – steroid class represented by cholesterol
4. Light absorbing pigment, electron carrier, signal molecules

Fatty acids: simplest lipids that are long-chain carboxylic acids

 Can contain up to 24 carbon atoms
 Most common fatty acids are even numbered (C16 and C18) – Ex.
palmitate and stereate
 Considered to be amphiphiles
o Polar head group – carboxylic acid
o Nonpolar tail – hydrocarbon chain

Can be saturated with hydrogens, unsaturated, or polyunsaturated

 In unsaturated fatty acids, double bond usually has the cis configuration

Free-Fatty acids in biological systems – they are usually esterified to glycerol

 Fats and oils in animals/plants are Triacylglycerols (acyl groups of three fatty acids are esterified to the
three hydroxyl groups of glycerol via a condensation reactions)

Triacylglycerols can aggregate in large globules.

 Serves as a storage depot for fatty acids that can be broken down to release
metabolic energy.
 Tendency to aggregate means that cells can store a large amount without it
interfering with other cellular activities in the aqueous cytosol.
Humans cannot make any double bonds past carbon 9; other organisms can produce Omega-3-Fatty Acids
 Omega-3-Fatty Acid: has a double bond starting three carbons from the methyl end.
 Essential Fatty Acids cannot be synthesized by humans and must be supplied in the diet
o Ex. EPA (C-20) and DHA (C-22) in cold-water fish; -linoleic acids
in plants
 Linoleic and linolenic fatty acids are precursors for longer chain Omega-
3-Fatty Acids
o Diets without fish oil acquire -linoleic acid from plants and lengthen the chain from the carboxylic

Why are essential fatty acids and omega-3-fatty acids important?

 Omega-3-fatty acids are essential for normal human growth
 EPA linked to decreased risk of cardiovascular disease – omega-3 compete with omega-6 for enzymes that
convert fatty acids to signaling molecules
o Omega-6 derivatives are stronger triggers of inflammation – atheroscelerosis
o The relative amounts of omega-3 and omega-6 are more important than the absolute amount of
 DHA (C-22): is abundant in the brain and retina and its concentration decreases with age.
o Protects neural tissues from damage following a stroke, but cannot reverse cognitive decline
associated with Alzheimer’s disease.

Glycerophospholipids: contain a glycerol backbone with fatty acyl groups

esterified at position 1 and 2, and a phosphate head group at position 3.
 Major lipids of the biological membrane
 Considered amphipathic: hydrophobic tails attached to polar/charged head

Phospholipases are enzymes that hydrolyzes glycerophospholipids to release

acyl chains
 Phospholipids are cleaved at specific
 For degrading lipids
 Act as signaling molecules
inside/between cells

Sphingolipids: amphipathic membrane lipid

molecules that use sphingosine as a backbone
 Sphingomyelins – have phosphocholine or
phosphoethanolamine head groups
 In a sphingolipid, a second fatty acyl group is
attached via an amide bond to the serine nitrogen

Glycolipids are sphingolipids with one or more

carbohydrate groups
 Cerebroside – monosaccharide head group
 Ganglioside – oligosaccharide head group
Cholesterol: 27 carbon, four-ring molecule found in biological
 Important component in biological membranes
 Metabolic precursor of steroid hormones (such as estrogen and
testosterone) Reactive Portion
 Has an amphiphilic character
 It is an isoprenoid (terpenoids) – made out
of a 5-carbon unit, isoprene
 Can be found in animal tissues

Ubiquitin is an isoprenoid derivative (functions in

electron transport)

Phytosterols: found in plants; main consituents in plant membrane structure

 Ex. stigmasterol, -sitosterol, campesterol
 Constituent in plant membrane structure
 Are found to decrease the level of LDL in the blood
 -sitosterol is found to be against enlargement of the prostate – may inhibit prostate
cancer cells

Clinical Connection Lipid Vitamins A, D, E, and K (isoprenoid derivatives)

I. Vitamin A (Retinol) – derived from (orange pigment in carrots)

 Role in light reception in the eye; retinol is oxidized to retinal (an
aldehyde) which functions as a light receptor’
 Light causes retinal to isomerize, causing an impulse through the optic
 Retinoic acid acts like a hormone by stimulating tissue repair

II. Vitamin D (Steroid Derivative)

 Vitamin D2: derived from plants
 Vitamin D3: derived from endogenously produced cholesterol
 UV light is required for the formation of Vitamin D2 and D3
 Enzymes in the liver and kidney convert Vitamin D to its active form,
stimulating calcium absorption in the intestine
o High [Ca2+] in bloodstream promotes Ca2+ deposition in bones and

III. Vitamin E (-Tocopherol): hydrophobic molecule incorporated into cell membranes

 Reacts with free radicals generated during oxidative
 Stems from activity as a regulatory molecule that
suppresses free-radical formation by inhibiting
production or activation of oxidative enzymes
 Binds to biological membranes
IV. Vitamin K (Phylloquinone): participates in enzymatic carboxylation of Glu residues in proteins involved in
blood coagulation
 Vitamin K deficiency: prevents Glu carboxylation, inhibiting normal
function of coagulating proteins causing excessive bleeding
 Half the daily uptake of Vitamin K is supplied by intestinal bacteria

Excess vitamin A,D,E,K accumulate in fatty tissues.

 Excess vitamin D: kidney stones and abnormal calcification of soft tissues
 High levels of vitamin A: produce symptoms such as birth defects

 Waxes (produced by plants on surfaces of leaves and fruits)
 In humans, derivative of Arachidonate (C-20): signaling molecules that regulate blood pressure, pain and
 All molecules derived from isoprene.
 Provides odors and colors in plants.

Capsaicin: compound found in chili peppers

 Hydrophobicity explains why it cannot be washed away with water.
 Pain Reliever: activates receptors on neurons that sense pain and heat
and overwhelms the receptor, preventing neurons from receiving pain
Chapter 17 – Lipid Metabolism
Atherosclerosis slow progressive disease that begins with the accumulation of lipids in the walls of large blood
vessels (1/2 of US deaths)
 Trapped lipids initiate inflammation by triggering the production of chemical signals that attract leukocytes
and macrophages – cells engorge themselves by taking up accumulated lipids, recruiting more
macrophages leading to continued inflammation
 Damaged vessel wall forms a plaque with a core cholesterol, cholesteryl
esters and dead macrophages that are surrounded by smooth muscle cells
that can undergo calcification

More energy from fats; cardiac muscle use 95% energy from fatty acids
There is a higher energy of fatty acids because there are more reduced; more
oxidation; more ATP

 Bile salts emulsify fatty acids (increase SA); more

exposed to pancreatic lipases; break up micelles
 The products of emulsification are shuttled to brush
border in bile micelles and then absorbed by
enterocytes; bile reabsorbed by small intestine and
transported to the liver
 Repackaging happens in intestinal mucosa
 Repackaged in lipoproteins
 Chylomicrons transport dietary TAG
 Lipoprotein lipases- breakdown lipoproteins
 TAGs reabsorbed by muscle or adipocytes

What is the source of lipids that accumulate in vessel walls? They are deposited by the lipoprotein, LDL.

Lipoproteins are made of phospholipids, can travel through bloodstream because of polar heads and proteins
 Lipids are in the core
 The density of lipoproteins are dependent on protein content and also differ in diameter
o Chylomicrons are the largest (least dense)
o HDL are the smallest (densest)
 Lipoproteins are the primary form of circulating lipid
 Dietary lipids travel from the intestine to other tissues as chylomicrons via the lymphatic system
The main function of chylomicrons is to transport dietary TAG to adipose tissue and cholesterol to the liver
 The liver repackages the cholesterol and other lipids (TAG, phospholipids, cholesteryl esters) into VLDL
 VLDL give up TAG to the tissues, becoming smaller, denser, and richer in cholesterol and cholesteryl esters
 IDL becomes LDL

LDL is considered bad because

it has more TAG and cholesterol
compared to HDL (good
 High concentration of
circulating LDL measured as
serum cholesterol; major
factor in atherosclerosis
Atherosclerosis less likely to occur in individuals who consume low-cholesterol diets and who have high levels
of HDL
Primary function of HDL is to transport body’s excess cholesterol back to the liver (Liver packages lipids)
 Counteracts the atherogenic tendencies of LDL

HDL scrapes cholesterol from tissue to liver; signal to liver

 Cholesterol is negative feedback inhibitor of cholesterol
Serum albumin- 50% of protein in blood stream, transports fatty
acids, hormones

Fatty Acid oxidation

Lipoproteins carry TAGs to tissues where hydrolysis releases their fatty acids from the glycerol backbone
 The hydrolysis of TAG occurs extracellularly, catalyzed by lipoprotein lipase
TAG that are stored in adipose tissue are mobilized by an intra-cellular hormone sensitive lipase
 Mobilized fatty acids travel through bloodstream bound to albumin
 (66 kD protein that accounts for half the serum protein, bind metal ions and hormones, serves as an all-
purpose transport protein)
The concentration of free fatty acids in the body is very low because these molecules are detergents and can
disrupt cell membranes
High glucose = insulin
To be oxidatively degraded, a fatty acid must be activated
Low glucose = glucagon, and epinephrine
TAG activation requires 2 ATP equivalents, so we have to subtract 2 to achieve ne
 Activated fatty acids are acylated to CoA
 TAG activation occurs in the cytosol

Reaction is driven by ATP hydrolysis

The reaction is almost in equilibrium; to make it spontaneous, phospohanhydride bond is broken, releases E
Catalyzed by Acyl-CoA synthetase
Most cells contain a set of Acyl-CoA synthetases specific for fatty acids that are short, medium long, very

 Acyl-CoA requires a transporter into mitochondria, carnitine acyl transferase (inhibited by malonyl-
 Cytosol into the mitochondria
Beta Oxidation
Beta oxidation is a spiral pathway that has 4 enzymatic steps;
 Each saturated fatty acid produces 1 NADH, 1 FADH2 (QH2), 1 Acetyl-CoA, an Acyl-CoA that is 2 C shorter
 Each Acetyl-CoA produced goes into the TCA cycle where 3 NADH, 1 FADH2, 1 ATP are produced
 Beta oxidation feeds directly into the ETC to produce ATP (NADH, FADH2 go into the ETC)

Total ATP produced is 14 ATP from one round of beta oxidation (12 ATP net because 2 consumed in activated
in cytosol)
 Rounds of beta oxidation: #C / 2 – 1
 Beta oxidation occurs on the beta C, third carbon; the two carbons receding the beta C are removed during
each round
The steps of a beta oxidation round:
 Oxidation (produce FADH2)
 Hydration (enoyl-CoA hydratase)
 Oxidation (produce NADH) (Dehydrogenation)
 Cleavage (2 C at a time)

The first step creates a trans 2, 3 double bond

Enoyl-CoA hydratase requires cis 2, 3 double bond to be
activated; unsaturated fatty acids have to go through extra
steps to achieve this configuration; which is why they are
less fattening
Degradation of unsaturated fatty acids requires isomerization and
Common fatty acids (oleate and linoleate) contain cis double bonds – obstacle to
enzymes that catalyze B-oxidation
 Linoleate has an acyl-CoA that has 3,4 double bond – cis enoyl-CoA
 Enoyl-CoA hydratase (2nd step of B-oxidation) recognizes only trans.
 Enoyl-CoA isomerase converts cis-3,4 to trans-2,3 double bond for B-oxidation
to continue
 Isomerization skips step 1 (1 less FADH2)
 Reduction consumes NADPH = -1 NADH
 4 total ATP less than saturated fatty acids per round of beta oxidation

This is why unsaturated fatty acids are not as fattening, because they do not
produce as much ATP
 Compounds with double bonds are more oxidized than saturated
compounds, so less energy is released in converting them to CO2
 The enoyl-CoA isomerase bypasses the QH2 producing acyl
dehydrogenase step (1.5 fewer ATP)
 NADPH dependent reductase consumes 2.5 consumes 2.5 ATP

Oxidation of odd-chain fatty acids yields propionyl-CoA

Most fatty acids are even numbered Carbon chains because synthesizing fatty
acids occurs by adding 2 C at a time (opposite of beta oxidation)
 The final round of beta oxidation of odd numbered fatty acids yields the
three carbon compound propionyl-CoA rather than Acetyl-CoA
The complete catabolism of carbons derived from propionyl-CoA requires
that succinyl-CoA be converted to pyruvate and then to Acetyl-CoA, which
enters the TCA cycle
Propionyl-CoA  Acetyl CoA
Methylmalonyl-CoA mutase, which catalyzes step three is an unusual enzyme
because it mediates a rearrangement of carbon atoms
 Requires a prosthetic group derived from B12 (cobalamin)

Some fatty acid oxidation occurs in the peroxisomes

Peroxisomes are organelles that are bound by a single membrane
 Peroxisome produce H2O2; which is broken down to water and O2
Longer fatty acids are oxidized > C16 and branched fatty acids as well in peroxisomes
 In plants, all fatty acid oxidation occurs in peroxisomes and glyoxysomes

Electrons removed from Acyl-CoA are transferred from FADH2 to H2O2 instead of ubiquinone
 FADH2 does not form ATP
 Fatty acid oxidation is different than in peroxisomes
 Peroxisome catalase breaks down hydrogen peroxide
 The peroxisome serves as a chain shortening system because it has low affinity for short chains and high
specificity for long chains – peroxisomes break down fatty acids unrecognized by mitochondrial enzymes
 Peroxisomes also degrade branched fatty acids (such as phytanate, which is from chlorophyll)
 Phytanate must be degraded by the peroxisome
because because the methyl group at C3 prevents
dehydrogenation by 3—hydroxyacyl-CoA
dehydrogenase (step three of standard beta oxidation)
 Deficiency in phytanate-degrading enzymes results in Refsum’s disease, a degenerative neural disorder
characterized by the accumulation of phytanate in the tissues

Fatty Acid Synthesis

Fatty acyl groups are built and degraded two carbons at a time
The pathways for fatty acid synthesis and degradation must differ for thermodynamic reasons
 Fatty oxidation is a thermodynamically favorable process; reversing the steps of the pathway would not be
energetically favorable
Beta oxidation takes place in the mitochondrial matrix, while fatty acid synthesis takes place in the cytosol
 In beta oxidation, the acyl group is attached to coenzyme A
 In fatty acid synthesis, the acyl chain is bound by an acyl-carrier protein ACP
 Beta oxidation requires two ATP to activate the acyl group
 Fatty acid synthesis consumes one ATP and 2 NADPH per round (Addition of 2C)
 B7 (biotin) is the precursor of Acyl-Carrier
protein and CoA
 NADPH is the source of reducing power

Acyl chains are carried by:

 CoA in fatty acid oxidation
 Acyl-carrier protein in fatty acid synthesis

Acetyl-CoA carboxylase catalyzes the first step of fatty acid synthesis

Acetyl-CoA is the starting material for fatty acid synthesis (generated in
mitochondria by the PDH)
 Acetyl-CoA cannot exit the cytosol for biosynthetic reactions
 Transport of acetyl groups to the cytosol involves the Citrate
Transport System
 ATP is consumed in the ATP-citrate lyase reaction to drive formation
of the thioester bond
The first step of fatty acid synthesis is the carboxylation of acetyl-CoA
(carried out by Acetyl-CoA carboxylase)
 Acetyl-CoA carboxylase is the regulatory enzyme (rate-controlling)
 CO2 (as bicarbonate) is “activated” by its attachment to a biotin (B7) prosthetic group (Uses 1 ATP)

Malonyl-CoA is the donor of the 2C acetyl units

that are used to build a fatty acid

 Biotin is the carrier of CO2 which is added to

Acetyl-CoA to make malonyl-CoA
 We use the 3C malonyl-CoA because we need
the other C for CO2 (released; E released)
Fatty acid synthase catalyzes seven reactions
The protein carrying out fatty acid synthesis is
multifunctional enzyme made of two identical
 Fatty acid synthase has six active sites to
carry out seven discrete reaction
 In plants and bacteria, the reaction is
catalyzed by separate polypeptides; same
 Reactions 1 & 2 are transacetylation reactions – prime/load the
enzyme w the reactants for the condensation reaction
 The condensation reaction combines Malonyl-ACP and Acetyl-Cys to
make Acetoacetyl-ACP
 1 ATP inputted to make malonyl-CoA; condensation reaction is
energized form E released from CO2 release
 Malonyl-CoA is a two-carbon donor (CO2 released = E released)
 Growth of the acyl chain (like chain shortening in beta oxidation) occurs at the thioester end of the
The NADH required for reduction steps (4 and 6) of FAS is supplied by the Pentose Phosphate Pathway
 The synthesis of palmitate requires
production of 7 malonyl-CoA at cost of 7
 Consumes 14 NADPH (= 35 ATP) – total
cost of 42 ATP

Packaging several enzyme activities into one multifunctional protein like mammalian fatty acid synthase allows
the enzymes to be synthesized and controlled in a coordinated fashion
 The product of one reaction can quickly diffuse to the next acid site

Other enzymes elongate and desaturate newly synthesized fatty acids

Sphingolipids that contain C22 and C24 fatty acyl groups are generated by enzymes known as elongases
 They extend the C16 fatty acid produced by fatty acid synthase

These reactions occur in the endoplasmic reticulum or mitochondria

 In ER: malonyl-CoA is the acetyl-group donor and are chemically similar to those of fatty acid synthase
 In mitochondria: fatty acids are elongated by reaction that closely resemble the reversal of beta-ox, but

Desaturases introduce double bonds into saturated fatty acids; take place in
the ER
 Electrons removed in dehydrogenation of fatty acid are transferred to O2 to
produce H2O
 Most common unsaturated fatty acids in animals are palmitoleate (C16)
and oleate (C18); both with one cis double bond at the 9,10 position
 Trans fatty acids are relatively rare in plants and animals
Elongation can follow desaturation (and vice versa) – animals can synthesize a variety of fatty acids ,
HOWEVER, mammals cannot introduce double bonds at positions beyond C9
 Cannot synthesize fatty acids such as linoleate and linolenate (are precursors of arachidonate (C20) and
other lipids with specialized biological activities)
 Mammals must obtain linoleate and linolenate from their diet
 Essential fatty acids are abundant in fish and plant oils
 Omega-3 fatty acids- unsaturated fatty acids with a double bond three carbons from the end, may have
health benefits (deficiency from very-lowfat diet may cause slow growth/wound healing)

Fats, Diet and Heat Disease (Biochemistry note)

How do dietary lipids influence serum lipid levels?
 Diets rich in saturated fats have increased blood cholesterol (LDL)
 Diets rich in unsaturated vegetable oils replaced the saturated fats have the opposite effects
Production of semisolid margarine from liquid plant oils (TAGs containing unsaturated fatty acids) often
includes a hydrogenation step to chemically saturate the carbons of the fatty acyl chains
 In the hydrogenation process, some of the original cis double bonds are converted to trans
 Trans fatty acids have a tendency to increase LDL levels and decrease HDL levels
 Scientists still do not fully understand how the consumption of specific fatty acids (saturated or
unsaturated, cis or trans) influences lipoprotein metabolism
 A consequence of low-fat diets is that individuals consume relatively more carbs

Regulation of Fatty Acid Synthesis

Under conditions of high metabolic fuel, the products of carb and amino acid catabolism are directed toward
fatty acid synthesis (resulting fatty acids are stored as TAGs)
 Rate of FAS is regulated by Acetyl-CoA carboxylase, which catalyzes the first step of FAS
 Inhibited by palmitoyl-CoA
 Activated by citrate
 Is subject to allosteric regulation by hormone
simulated phosphorylation and dephosphorylation
 High Acyl-CoA and citrate activates FAS
 Citrate activates FAS

Malonyl-CoA is critical for preventing the wasteful

simultaneous activity of fatty acid synthesis and fatty
acid oxidation
 Is the source of acetyl groups that are incorporated
into fatty acids
 Also blocks beta-oxidation by inhibiting carnitine
acyltransferase (involved in shuttling acyl groups
from the cytosol into the mitochondria)
 Consequently, when fatty acid synthesis is under
way, no acyl groups are transported into the
mitochondria for oxidation
 FAS inhibited by fatty acid
Clinical Connection: inhibitors of FAS
Triclosan was believed to act as a general microbicide (microbicide are effective because it is difficult for
bacteria to evolve specific resistance mechanism)
 Triclosan operates more like an antibiotic with a specific
biochemical target; enoyl-ACP reductase, which catalyzes
step 6 of FAS
 In the active site, one of the phenyl rings of triclosan,
whose structure mimics the structure of the reaction
intermediate, stacks on top of the nicotinamide ring of the
NADH cofactor
 Triclosan also binds through van der Waals interactions and
hydrogen bonds with amino acid residues in the active site

Isoniazid is used against M. tuberculosis

 Isoniazid is oxidized and the reaction product combines with NAD+ to generate a compound that inhibits
one of the cell’s enoyl-ACP reductases (the target enzyme is specific for extremely long-chain fatty acids
Some fungal species are susceptible to cerulenin, which inhibits its 3-ketoacyl-ACP synthase (step 3 of FAS) by
blocking the reaction of malonyl-ACP (the condensation step)
 Cerulenin is also effective against M. tuberculosis, inhibiting the production of long-chain fatty acids
required for cell-wall synthesis
 The drug contains a reactive epoxide group; reacts irreversibly with the enzyme’s active-site Cys residue
forming a C2-S covalent bond; the hydrocarbon tail occupies the site that would normally accommodate
the growing fatty acyl chain

Acetyl-CoA can be converted to ketone bodies

During a fast (when glucose is unavailable) tissues depend on fatty acids released from stored triacylglycerols,
but the brain does not burn fatty acids (pass poorly through blood-brain barrier)
 Liver produces ketone bodies to supplement gluconeogenesis

Acetoacetate and 3-hydroxybutyrate (ketone bodies) are synthesized from Acetyl-CoA in liver mitochondria
by the process, Ketogenesis.
 Because it uses fatty acid-derived acetyl
groups, it helps spare amino acids that would
be diverted to gluconeogenesis
 Ketone body synthesis occurs in the liver
 Liver does not use ketone bodies, however
they donate synthesized ketone bodies,
missing 3-ketoacyl-CoA transferase
 Assembly of ketone bodies is somewhat
reminiscent of the synthesis of fatty acids or
the oxidation of fatty acids in two-carbon steps
Ketone bodies are transported in the bloodstream without specialized lipoproteins because they are small and
water-soluble – easily pass through CNS
 High ketogenic activity (such as in diabetes), ketone bodies are produced faster than they are consumed.
 Excess acetoacetate breaks down to acetone - sweet breath is characteristic of acetone
 Ketone bodies are acidic, leading to a drop in blood pH – ketoacidosis
Ketone bodies produced by the liver are used by other tissues as metabolic fuels after being converted back to
 The liver itself cannot catabolize ketone bodies because it lacks 3-ketoacyl-CoA transferase

Synthesis of Other Lipids

TAGs and phospholipids are built from Acyl-CoA groups
TAGS are synthesized by attaching fatty acyl groups to a
glycerol backbone derived from phosphorylated glycerol
or from glycolytic intermediates
 Synthetase requires ATP
 Synthase does not require ATP
 Ex. Dihydroxyacetone phosphate

Fatty acyl groups are activated to CoA thioesters in an ATP-dep.

manner catalyzed by Acyl-CoA synthetase (which also activates
fatty acids for oxidation)
 Acyl transferase (that adds fatty acids to the glycerol
backbone) are not highly specific, but human TAGs usually
contain palmitate at C1 and unsaturated oleate at C2

The TAG biosynthetic pathway also provides the precursors for

 These amphipathic phospholipids are synthesized form phosphatidate (phosphorylated diacylglycerol) or
diacylglycerol by pathways that include an activating step in which cytidine triphosphate (CTP) is cleaved
(in some cases, the phospholipid head group is activates; in other cases, the lipid tail portion is activated)
 CTP is required for energy in order to activate the reactant

The fundamental similarity between phospholipid and triacylglycerol is the glycerol backbone

 Phosphatidylserine is synthesized from phophatidylethanolamine by a head group exchange reaction in

which serine displaces the ethanolamine head group
 Phosphatidylinositol synthesis: the diacylglycerol component is activated, rather than the head group, so
the inositol head group adds to CDP diacylglycerol
Glycerophospholipids (and sphingolipids) are components of cellular membranes
 New membranes are formed by inserting protein and lipids into preexisting membranes, mainly in the ER
 Newly synthesized membrane components reach final cellular destinations via vesicles that budd of the ER
 May undergo remodeling through the action of phospholipase and acyltransferases that remove and
reattach different fatty acyl groups
Cholesterol Synthesis begins with Acetyl-CoA
Cholesterol molecules are built form two-carbon acetyl units
 The first steps of cholesterol synthesis resemble those of ketogenesis
 However, ketone bodies are synthesized in the mitochondria (and only in the liver), cholesterol is
synthesized in the cytosol
The reactions of cholesterol synthesis and ketogenesis diverge after
the production of HMG-CoA
 In ketogenesis, HMG-CoA is cleaved to produce acetoacetate
 In cholesterol synthesis, the thioester of HMG-COA is reduced to
alcohol, releasing mevalonate (6C)
Mevalonate is converted to the five-carbon isopentyl
pyrophosphate, an isoprene derivative.
 The isoprenoid derivative is the precursor of cholesterol
 As well as other isoprenoids such as ubiquinone, the farnesyl
group that is attached to some lipid-linked membrane proteins,
and pigments such as beta-carotene

In cholesterol synthesis, six isoprene units condense to form squalene

 Cyclization of squalene leads to a structure with four rings,
resembling cholesterol
 A total of 21 reactions are required to convert squalene to
cholesterol (NADH or NADPH is required for several steps)
 The rate-determining step of cholesterol synthesis and the major
control point is the conversion of HMG-CoA to mevalonate by
HMG-CoA reductase (one of the most highly regulated enzymes known)

Synthetic inhibitors known as statins bind extremely tightly to HMG-CoA reductase

 All the statins have and HMG-like group that acts as a competitive inhibitor of HMG-CoA binding to the
 The rigid hydrophobic groups also prevent the enzyme from forming a structure that would accommodate
the pantothenate moiety of CoA
 Physiological effects of statins are to lower serum cholesterol levels by blocking mevalonate synthesis

Cells must obtain cholesterol from circulating lipoproteins, but since mevalonate is also the precursor of
other isoprenoids such as ubiquinone, the long-term use of statins can have negative side effects
 Cholesterol affects membrane fluidity
 Cholesterol packaged in lipoprotein

Cholesterol can be used in several

I. It can be incorporated into a cells membrane
II. It may be acylated to forma cholesteryl ester for storage or, in liver, for packaging in VLDL
III. It is a precursor of steroid hormones such as testosterone and estrogen in the appropriate tissues
IV. It is a precursor of bile acids such as cholate
V. Bile salts remove cholesterol; serve as route for cholesterol exodus
Bile acids aid digestion by acting as detergents to solubilize dietary fats and make them more susceptible to
 Are mostly reabsorbed and recycled through liver for reuse, but some are excreted from the body – the
only route for cholesterol disposal
 The cholesterol contained in bile will occasionally aggregate into lumps in the gallbladder forming
Cells can synthesize cholesterol as well as obtain it from circulating
 When LDL proteins dock with LDL receptor on the cell surface, the
lipoprotein-receptor complex undergoes endocytosis – the
lipoprotein is degraded and cholesterol enters the cytosol
 Familial hypercholesterolemia: genetic defect in the LDL receptor – the cells of homozygotes are unable
to take up LDL, the concentration of serum cholesterol is about 3 times higher than normal

High-density lipoproteins (HDL) are essential for removing excess cholesterol from cells
 The efflux of cholesterol requires the close juxtaposition of the cell membrane and an HDL particle as well
as specific cell-surface proteins
 Such as the ABC transporter that acts as a flippase to move cholesterol
from the cytosolic leaflet to the extracellular leaflet, from which it can
diffuse into the HDL particle
o Defect in the gene for the transporter cause Tangier disease which is
characterized by accumulating of cholesterol in tissues and a high risk
of heart attack

Because cells do not break down cholesterol and because the accumulation of cholesterol is potentially toxic,
the body must coordinate cholesterol synthesis and transport among tissues
 Cholesterol shuts down its own synthesis by inhibiting the synthesis of enzymes such as HMG-CoA
 Cellular cholesterol also represses transcription of the gene for the LDL receptor
 Cholesterol metabolism in many cells is characterized by a balance between influx and efflux
o Fatty acid metabolism has two opposing pathways of synthesis and degradation that operate in
balance to meet the cell’s needs
Chapter 18 – Nitrogen Metabolism
Incorporation of ammonium ion into biological molecules
 80% of air is Nitrogen (N2) and must be fixed (Fixed Nitrogen): Nitrite, Nitrate, and Ammonia

Nitrogenase: enzyme that carries out reduction of N2 into NH3 (Nitrogen Fixation)
 Is a metalloprotein containing Fe-S clusters and Fe-Mo Cofactor (Molybdenum, Iron, Sulfur)
 Ammonia exists in protonated form (NH4+)
 Nitrogen fixing bacteria such as marine cyanobacteria and bacteria colonize root nodules of legumes

Nitrogen Fixation – conversion of N2 to NH4+ using nitrogenase

 Consumes a lot of ATP
 Ferredoxin, a strong reducing agent donates 8 electrons

Nitrate/Nitrite reductase catalyzes nitrate to nitrite to ammonia

Bacteria produce nitrate via nitrification

Other organisms convert nitrate to N2 via denitrification

Ammonia assimilation via Glutamine Synthetase and Glutamate

Glutamate and glutamine are at higher concentrations than other amino acids – carriers
of amino groups
 Glutamine synthetase: produces glutamine from glutamate (Synthetase = uses ATP)
 Glutamate synthase: produces glutamate from glutamine (Synthase = does not use ATP)

Glutamine synthetase: glutamate is phosphorylated via ATP,

then ammonia reacts with the intermediate and displaces Pi
to produce Glutamine

Glutamate synthase: nitrogen is assimilated into an

intermediate from the TCA cycle to produce Glu
 The source of nitrogen in Glutamate is Glutamine
 Glutamine is deaminated to produce a second Glutamate

The combined action of the two enzymes assimilates fixed nitrogen

(NH4+) into -ketoglutarate to produce Glutamate
 Mammals lack glutamate synthase, but [Glu] are relatively high due
to other reactions
Transamination: moves amino groups between compounds – is a reversible
 Transaminase (or aminotransferase) catalyzes the transfer of an amino
group to a -ketoacid
o Use a prosthetic cofactor, Pyridoxal-5’-phosphate (PLP) – a
prosthetic group that the amino group is
transiently attached to
o PLP is derived from pyridoxine (Vitamin B6)
o Amino group transfer occurs through a Lys
Residue (Schiff base linkage)

Amino Acid Biosynthesis

Amino Acids are synthesized from intermediates of glycolysis and the citric acid cycle

Ultimate sources of essential amino acids (must be obtained from food) are from plants and
 Nonessential Amino Acids (can be synthesized): Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, Pro,
Ser, Tyr
 Essential Amino Acids (must be obtained): His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val

Several amino acids are easily synthesized from common intermediates

 Alanine produced from Pyruvate

 Aspartate produced from Oxaloacetate
 Glutamate from -Ketoglutarate

Tetrahydrofolate: a carrier of one-carbon units in several reactions of amino acids and nucleotide metabolism.
 Mammals cannot synthesize folate and must be
obtained from diet
o Requirement for folate increases during
first few weeks of pregnancy for
development of fetal nervous system;
prevents neural tube defects such as
spina bifida
 Serine is derived from 3-phosphoglycerate (3-
PG, from glycolysis) and can give rise to Glycine
via serine hydroxymethyl transferase
Amino acids with sulfur, branched chains, or aromatic groups are more difficult to synthesize
: Amino acids include Met, Cys, Tyr, Trp, His, Ile
Ser  Cysteine

(Bacteria) (Humans)

High levels of homocysteine in blood is associated with homocystinuria (excess homocysteine is excrete)
 Individuals develop atherosclerosis in children, homocysteine directly damages blood vessel walls

Apartate is a precursor for Methionine, Threonine and Lysine

 Branched chain AA (Valine, Leucine and Isoleucine) are synthesized by pathways with pyruvate as a
starting substrate and requires a step catalyzed by transaminase and glutamate as a substrate

The synthesis for the Aromatic Amino Acids (Phenylalanine, Tyrosine, and Tyrptophan) begin with
condensation of Phosphoenolpyruvate (3-C, glycolysis) and Erythose-4-phosphate (4-C, PPP)
 The 7 carbon reaction produce cyclizes and is modified to become
 Animals do no synthesize chorismate  pathway is a target for
agents that inhibit plant metabolism without affecting animals

Phenylalanine and Tyrosine are derived from chorismate via diverging

 In humans, tyrosine is produced by hydroxylating phenylalanine
(Tyr is not an essential amino acid)

Tryptophan Synthase: catalyzes final two steps of Trp synthesis

 Active sites in adjacent subunits are connected by a tunnel that
accommodates indole
 Channeling: movement of reactant between two active sites – increases rate of metabolic process by
preventing loss of intermediates

Histidine is not formed from carbohydrate metabolites, rather is formed by ATP

providing one nitrogen and one carbon; and glutamate and glutamine donating
two N and remaining 5 carbons from 5-phosphoribosyl pyrophosphate (PRPP)

Box 18-B – Glyphosate, the most popular herbicide

Glycine phosphonate (glyphosphate) competes with PEP in pathway leading to chorismate
 Plants cannot manufacture aromatic AA without chorismate – glyphosate is an herbicide
 Glyphosate not directly absorbed to plant binds tight to soil particles and is broken down by bacteria
 Has less potential to contaminate water supply
 For effectiveness: glyphosate must enter plant tissues, therefore is packaged in a surfactant (amphiphilic)
that helps penetrate waxy coatings of leaves
Farmers plant genetically engineered glyphosate-resistant crops (corn, soybean, cotton) that express a
bacterial version of the enzyme that uses PEP but not inhibited by glyphosate
Amino acids are precursors of some signaling molecules
Amino acids can be precursors to neurotransmitters
 Common neurotransmitters: glycine, glutamate, and -aminobutyrate (GABA, a glutamate derivate)

Catecholamines: amino acid neurotransmitters that resemble catechol

 Ex. Tyrosine gives rise to dopamine, norepinephrine, and epinephrine

Tryptophan (a catecholamine) is precursor of serotonin, which is a precursor to


Box 18-C Nitric Oxide

Arginine is a precursor for the signaling molecule, free-radical nitric oxide (NO) – relaxation factor that elicits
 NO is synthesized by nitric oxide synthase (has cofactors FMN, FAD, tetrahydrobiopterin, & a heme group)
 At low concentrations, it induces blood vessel dilation
 At high concentrations (along with oxygen radicals), kills pathogens
Cannot be stockpiled for later release; diffuses into cells – no cell-surface receptor or degradative enzyme
 Is produced when and where it is needed

Nucleotide Biosynthesis
Nucleotides are synthesized from precursors that include amino acids
*Why don’t humans require purine and pyrimidines in their diet?

Purine nucleotide synthesis

Purines nucleotides (AdenineMP and GuanosineMP) are synthesized by building purine base onto ribose-5-
 First step: Ribose-5-phosphate into 5-phosphoribosyl pyrophosphate (also precursor to histidine)
 Next ten steps require Glu, Gly, Asp, bicarbonate and a
formyl from tetrahydrofolate to produce a nucleotide
product: Inosine Monophosphate (IMP)

IMP is the substrate for the pathway that yield AMP and GMP
 GTP participates in AMP synthesis: amino group from
aspartate is transferred to purine
 ATP participates in GMP synthesis: glutamate is source of
amino group

High concentrations of ATP  GMP Production

High concentrations of GTP  AMP Production

*Regulated via Feedback inhibition

Pyrimidine nucleotide synthesis
*What are the requirements for pyrimidine nucleotide synthesis?

Pyrimidine nucleotides (UracilTP and CytosineTP) are synthesized as a base that

attaches to 5-phosphoribsyl pyrophosphate
 Six-step pathway yields Uridine Monophosphate (UMP) requires Glu, Asp, and

UMP is phosphorylated into UDP and then to UTP, then CTP

synthase catalyzes amination of UTP to CTP (glutamine is
the donor)
 Regulated via Feedback Inhibition by UMP, UDP and
 ATP activates the enzyme

Ribonucleotide reductase: converts ribonucleotides to deoxyribonucleotides

 RNA has catalytic activity due to the OH on Carbon 2
o Ribose: OH on C2
o Deoxyribose: H on C2 – is more stable than ribose; DNA carries genetic information
 Each of the four nucleoside triphosphates (NTPs) are converted to their diphosphate (NDP) form,
ribonucleotide reductase replaces the 2’ OH group, the resulting deoxynucleoside diphosphate (dNP) is
phosphorylated to produce the corresponding triphosphate (dNTP)

Thyrimidine nucleotides are produced by methylation

*Explain the importance of thymidylate synthase and dihydrofolate reductase reactions

Ribonucleotide reductase, followed by kinase phosphorylation generates dATP, dCTP, dGTP, and dUTP
 dUTP is not used for DNA synthesis  it is rapidly converted to thymine nucleotides
 Thymine nucleotides help prevent accidental incorporation of uracil into DNA

dUTP is hydrolyzed to dUMP, then thymidylate synthase adds methyl to dUMP to produce dTMP using
methylene-tetrahydrofolate as one-carbon donor
 Main source of methylene-tetrahydrofolate: serine hydroxymethyltransferase reaction (serine to glycine)
 Thymidylate synthase oxidizes tetrahydrofolate to dihydrofolate
 Dihydrofolate reductase (NADPH-dependent enzyme) then regenerates tetrahydrofolate cofactor
 dTMP is then phosphorylated to dTTP – substrate for DNA polymerase

In cancer cells that undergo rapid cell division, thymidylate synthase and dihydrofolate reductase are highly
 Compounds that inhibit these reactions are anti-cancer agents
 Methoxetrate (an antifolate) is a competitive inhibitor of dihydrofolate reductase because they compete
for binding
o In presence of methoxetrate, Cancer cells cannot regenerate tetrahydrofolate required for dTMP
production and cell dies
Nucleotide degradation produces uric acid or amino acids
Nucleotides from food or from synthesis by cells can be broken down to release ribose groups and
purine/pyrimidine that can:
 Be further catabolized and excreted (purines)
 Or be used as metabolic fuel (pyrimidines)
 At some points in degradation pathways, intermediates can be redirected towards synthesis of new
nucleotides by salvage pathways

Degradation of nucleoside phosphate

 Begins with dephosphorylation to produces a nucleoside
 Phosphorylase breaks glycosidic bond between the base and
ribose in a nucleoside

The phosphorylated ribose can be catabolized or salvaged via conversion to 5-PRPP for synthesis of another
nucleotide – fate of base depends on whether purine or pyrimidine:
I. Purine base catabolism: purines are eventually converted into Uric Acid
 Other organisms may further catabolize urate to generate Urea or
 May require deamination and oxidation – depends if base was adenine,
guanine or hypoxanthine
 Generates a waste produce that is excreted from the body

II. Pyrimidine base catabolism: cytosine, thymine, and uracil undergo deamination and reduction to open
the pyrimidine ring and further catabolism of these produces the amino acids:
 -alanine (from cytosine and uracil) or -aminoisobutyrate (from thymine)
 Contributes to the pool of metabolites for anabolic and catabolic processes

Amino Acid Catabolism

Amino acids are metabolic fuels that can be broken down to release free energy.
 Amino acids are major fuel for cells in the lining of the small intestine, which absorb dietary amino acids
and break down available Glu, Asp, and Gln supply
 The liver catabolize AA’s originating from the diet and from normal turnover of intracellular proteins.
o During periods of unavailable dietary AA’s (fasting), amino acids are mobilized from breakdown of
muscle tissue – 40% of total protein in the body.
o AA’s undergo transamination and carbon skeletons enter metabolic pathways (TCA cycle)
o However, there is not enough oxygen in liver for complete oxidation to CO2, therefore, amino acids
are partially oxidized to substrates for gluconeogenesis or

Classification of amino acids in terms of catabolism

 Glucogenic: giving rise to gluconeogenic precursors (TCA
 Ketogenic: giving rise to Acetyl-coA; used for ketogenesis or fatty
acid synthesis
Some amino acids are converted to gluconeogenic substrates via transamination
 Alanine to Pyruvate
 Aspartate to Oxaloacetate
 Glutamate to -Ketoglutarate

Cysteine can be converted into pyruvate that releases ammonia and sulfur

Threonine is both glucogenic and ketogenic because it is broken

down into acetyl-CoA and glycine
 Acetyl-CoA is a precursor for ketone bodies
 Glycine is potentially glucogenic if it is first converted to
serine vie serine hydromethyl transferase
o Glycine Cleavage System: major route for glycine disposal (multienzyme complex)

Degradation for Branched-chain AA’s (Val, Leu, Ile) and Aromatic AA’s (Phe, Tyr, and Trp) are more
 Ile yields succinyl-CoA and acetyl-CoA
 Leu yields acetyl-Aoa and acetoacetate (ketone body)
 Lysine yields Acetyl-CoA and acetoacetate (different pathway from branched AA’s)
 Met yields succinyl-CoA
 Phe, Tyr and Trp yield acetoacetate (Ketone Body)

Box 18-D Inborn Errors of Metabolism

Archibald Garrod: recognized link between genes and disease – studied alcaptonuria (urine turns black upon
exposure to air due to homogentisate, a product of tyrosine catabolism – homogentisate is excreted because
there is a missing or defective homogentisate dioxygenase enzyme)

Phenylketonuria (PKU) results from deficiency of phenylalanine

 Phenyalanine cannot be broken down but can undergo
transamination that results in an accumulation of phenylpyruvate
(-keto acid derivative) that is excreted in the urine

Nitrogen Disposal: The Urea Cycle

Excess supply of AA’s for cell’s immediate needs for protein synthesis or AA-consuming pathways causes
carbon skeletons to break down and the nitrogen disposed of
 All AA’s (except lys) can be deaminated but they are not eliminated from the body.

Approximately 80% of the body’s excess nitrogen is excreted in the form of Urea

*How are amino groups for amino acids incorporated into urea?
Glutamate supplies nitrogen to the urea cycle
Glutamate is deaminated to regenerate -ketoglutarate and release NH4+ via glutamate dehydrogenase
 Many transaminases use -ketoglutarate as amino group
acceptor – glutamate is abundant
 Only known enzyme that can use NAD+ or NADP+ as a cofactor
 Major route for feeding AA-derived amino groups into urea cycle
 Is subject to allosteric activation/inhibition

The starting substrate for the urea cycle is an activated molecule produced from the condensation of
bicarbonate and ammonia via carbamoyl phosphate synthetase

The Urea Cycle

The carbamoyl phosphate synthetase and glutamate dehydrogenase reaction are combined with the
reactions of the Urea cycle are combined
 Overall effect: transaminated AA’s donate amino groups via glutamate and aspartate to urea synthesis
 Amino acids can be disposed via two routes:

1. Carb
2. Glu

N-acetylglutamate is an activator of carbamoyl phosphate synthetase

 Rate of urea production is controlled by this enzyme and is allosterically synthesized by N-acetylglutamate
(produced from glutamate and acetyl-CoA)

Bacteria, fungi, and other organisms use urease to break down urea.
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