INTRODUCTION

Biokimia dan Biologi Molekuler (BBM)

(Biochemistry and Molecular Biology)
Biochemistry can be defined as the science of the chemical basis of life
(Gk bios "life").

The cell is the structural unit of living systems.

Thus, biochemistry can also be described as the science of the chemical
constituents of living cells and of the reactions and processes they
undergo.

By this definition, biochemistry encompasses large areas of cell biology,

molecular biology, and molecular genetics.
 The Aim of Biochemistry Is to Describe &
Explain, in Molecular Terms, All Chemical
Processes of Living Cells

The major objective of biochemistry is the complete understanding, at the molecular

level, of all of the chemical processes associated with living cells. To achieve this
objective, biochemists have sought to isolate the numerous molecules found in cells,
determine their structures, and analyze how they function:

Table 1. The Principal Methods and Preparations Used in Biochemical Labs:

1. Methods for Separating and Purifying Biomolecules:

- Salt fractionation,
- Chromatography (7)
- Electrophoresis
- Ultracentrifuge
2. Methods for Determining Biomolecular Structures:

- Elemental analysis
- UV, visible, infrared, and NMR spectroscopy
- Use of acid or alkaline hydrolysis to degrade the biomolecule
-Use of a battery of enzymes of known specificity to degrade biomolecule
- Mass spectrometry
- Specific sequencing methods (eg, for proteins and nucleic acids)
- X-ray crystallography

3. Preparations for Studying Biochemical Processes :

- Whole animal (transgenic animals and animals with gene knockouts)

- Isolated perfused organ
- Tissue slice
- Whole cells
- Homogenate
- Isolated cell organelles
- Subfractionation of organelles
- Purified metabolites and enzymes
- Isolated genes (PCR and site-directed mutagenesis )
 A Knowledge of Biochemistry Is Essential to All Life Sciences
- Genetic, Physiology, Immunology, Pharmacology and pharmacy,
- Toxicology, Pathology, Microbiology, Zoology, and Botany

These relationships are not surprising, because life as we know it

depends on biochemical reactions and processes. In fact, the old barriers among
the life sciences are breaking down, and biochemistry is increasingly becoming
their common language ( lingua franca )

A Reciprocal Relationship Between Biochemistry & Medicine Has

Stimulated Mutual Advances

The relationship between medicine and biochemistry

 The relationship between medicine and biochemistry has important implications
for the former. As long as medical treatment is firmly grounded in the knowledge of
biochemistry and other basic sciences, the practice of medicine will have a rational
basis that can be adapted to accommodate new knowledge.

This contrasts with unorthodox health cults and at least some "alternative
medicine" practices that are often founded on little more than myth and wishful
thinking and generally lack any intellectual basis.

NORMAL BIOCHEMICAL PROCESSES ARE THE BASIS OF HEALTH

Biochemical Research Has Impact on Nutrition & Preventive Medicine

Most & Perhaps All Diseases Have a Biochemical Basis
Summary
Proteins and Amino Acids
Introduction: Why Proteins are very Important

Life on Earth depends on the biochemistry of two classes of

macromolecule: nucleic acids and proteins. Nucleic acids carry the genetic
information which is passed from parents to progeny, and which provides
the blueprint for a particular living organism. That blueprint is essentially a
set of instructions for making proteins.

This obviously means that proteins are the key molecules in the
processes of life, and it is now known that virtually all the activities which
sustain living organisms are carried out by proteins.

Proteins are constructed on a simple pattern, but that pattern allows for
an almost endless diversity of structure and function.

Proteins are the most abundant biological macromolecules, occurring in

all
cells and all parts of cells.
The following are a few examples of proteins and what they do:

All proteins, whether from the most ancient lines of bacteria or from the most
complex forms of life, are constructed from the same set of 20 amino acids,
covalently linked in characteristic linear sequences.
 Proteins (from Greek - proteios - very important, 1838), are :
Biopolymers that consist of linear chains of amino acid residues.

All organism use the same 20 amino acids as buildig blocks for the assembly
protein molecules. These 20 amino acids are therefore often cited as the common
or standard amino acids.

Despitee limited number of amino acid types, the variations in the order in which
they are are connected and in the number of amino acids per protein, allow an
almost limetless variety of proteins.

There Are Four Levels of Protein Structure:

1. Primary structure is the sequence in which amino acids are covalently
connected to form the polypeptide chain.
2. Secondary structure - regions of regularly repeating conformations of the
peptide chain, such as -helices and -sheets
3.Tertiary structure - describes the shape of the fully folded polypeptide chain
4. Quaternary structure - arrangement of two or more polypeptide chains into
multisubunit molecule
Amino Acid and the Primary Structure of Proteins

General Structure of Amino Acids

Twenty common -amino acids have carboxyl

and amino groups bonded to the -carbon atom
A hydrogen atom and a side chain (R) are also
attached to the -carbon atom
 Zwitterionic form of amino acids

Under normal cellular conditions amino

acids are zwitterions (dipolar ions):
Amino group = -NH3+
Carboxyl group = -COO-

Prentice Hall c2002 Chapter 3 14

A. Aliphatic R Groups
B. Aromatic R Groups
C. Sulfur-Containing R Groups

Formation of cystine
D. Side Chains with Alcohol Groups
E. Basic R Groups

The side chain of histidine contains an imidazole ring substituent.

Lysine is a diamino acid, having a -amino group.
Arginine is the most basic of the 20 amino acids because its side-chain
guanidinium ion is protonated under all conditions normally found within a cell
F. Acidic R Groups and Their Amide Derivatives

Aspartate (Asp, D) and glutamate (Glu, E) are dicarboxylic amino acids and have
negatively charged hydrophilic side chains at pH 7.

Asparagine (Asn, N) and glutamine (Gln, Q) are the amides of aspartic acid and
glutamic acid, respectively. Although the side chains of asparagine and glutamine
are uncharged, these amino acids are highly polar and are often found on the
surfaces of proteins, where they can interact with water molecules.
Amino Acid Groups
Other Amino Acids and Amino Acid Derivatives

More than 200 different amino acids are found in living organisms. In addition to the standard
amino acids that are incorporated into proteins, all species contain a variety of L-amino acids
that are either precursors of the common amino acids or intermediates in other biochemical
pathways.
Examples are : homocysteine,
homoserine,
ornithine, and
citrulline .
S-Adenosylmethionine (SAM) is a common methyl donor in many biochemical
pathways .
Bacteria and fungi synthesize D-amino acids that are used in cell walls and in
complex peptide antibiotics such as actinomycin D.
Several common amino acids are chemically modified to produce biologically important
amines. These are synthesized by enzyme-catalyzed reactions that include decarboxylation
and deamination.

For example: Glutamate is converted to the neurotransmitter (GABA).

Histidine Histamin
Tyrosine Epinephrine (adrenaline)
Thyroxine (T4) , Triiodothyronine
(T3)
Prentice Hall c2002 Chapter 3 22
 Compounds derived from common amino acids.

Prentice Hall c2002 Chapter 3 23

21st amino acid & 22nd amino acid
A surprising discovery (1984) was that a 21st amino acid, Selenocysteine (which contains
selenium in place of the sulfur of cysteine), is incorporated into a few proteins in a wide variety
of species.
The 22nd amino acid is Pyrrolysine, found in some species of archaebacteria (2003).
Pyrrolysine is a modified form of lysine that is synthesized before being added to a growing
polypeptide chain by the translation machinery.

Selenocysteine and pyrrolysine have their own codons, AUG and UGA, respectively, which
is why they are considered as standard amino acids

Prentice Hall c2002 Chapter 3 24

Prentice Hall c2002 Chapter 3 25
Prentice Hall c2002 Chapter 3 26
 Stereochemistry of amino acids

19 of the 20 common amino acids have a

chiral -carbon atom (Gly does not)
Threonine and isoleucine have 2 chiral
carbons each (4 possible stereoisomers each)
Mirror image pairs of amino acids are
designated L (levo) and D (dextro)
Proteins are assembled from L-amino acids
(a few D-amino acids occur in nature)
 Peptide Bonds Link Amino Acids
in Proteins

Peptide bond - linkage between amino acids

is a secondary amide bond
Formed by condensation of the -carboxyl of
one amino acid with the -amino of another
amino acid (loss of H2O molecule)
Primary structure - linear sequence of
amino acids in a polypeptide or protein
Peptide bond between
two amino acids
 Polypeptide chain nomenclature

Amino acid residues compose peptide chains

Peptide chains are numbered from the N (amino)
terminus to the C (carboxyl) terminus
Example: (N) Gly-Arg-Phe-Ala-Lys (C)
(or GRFAK)
Formation of peptide bonds eliminates the
ionizable -carboxyl and -amino groups of the
free amino acids
Amino Acid Composition of Proteins

Amino acid analysis - determination of the

amino acid composition of a protein
Peptide bonds are cleaved by acid hydrolysis
(6M HCl, 110o, 16-72 hours)
Amino acids are separated
chromatographically and quantitated
Phenylisothiocyanate (PITC) used to derivatize
the amino acids prior to HPLC analysis
 Acid-catalyzed hydrolysis of a
peptide
 Resonance
structure
of the peptide
(a) Peptide
bondbond shown as a
C-N single bond
(b) Peptide bond shown as a
double bond
(c) Actual structure is a hybrid
of the two resonance
forms. Electrons are
delocalized over three
atoms: O, C, N

Prentice Hall c2002 Chapter 3 34

 Planar peptide groups in a
polypeptide chain

Rotation around C-N bond is restricted due to the

double-bond nature of the resonance hybrid form
Peptide groups (blue planes) are therefore planar

Prentice Hall c2002 Chapter 3 35

 Trans and cis conformations
of a peptide group
Nearly all peptide groups in proteins are
in the trans conformation

Prentice Hall c2002 Chapter 3 36

 Secondary Structure

The -helix

Prentice Hall c2002 Chapter 3 37

 -Sheets (a) parallel, (b)
antiparallel

Prentice Hall c2002 Chapter 3 38

Supersecondary Structures (MOTIFFS)

Prentice Hall c2002 Chapter 3 39

Domains

Many proteins are composed of several discrete, independently folded,

compact units
called domains. Domains may consist of combinations of motifs. The
size of a domain varies from as few as 25 to 30 amino acid residues to more
than 300.

Prentice Hall c2002 Chapter 3 40

Common domain folds.

Prentice Hall c2002 Chapter 3 41

Tertiary structure

Prentice Hall c2002 Chapter 3 42

Prentice Hall c2002 Quaternary structure. 43
 Common
motifs

Prentice Hall c2002 Chapter 3 44

 Common
domain folds

Prentice Hall c2002 Chapter 3 45

 Quaternary structure of
multidomain proteins

Prentice Hall c2002 Chapter 3 46

Binding of 2,3BPG to deoxyhemoglobin.

Prentice Hall c2002 Chapter 3 47

 Human antibody structure.

Prentice Hall c2002 Chapter 3 48

 Globular proteins

Usually water soluble, compact, roughly

spherical
Hydrophobic interior, hydrophilic surface
Globular proteins include enzymes,carrier
and regulatory proteins

Prentice Hall c2002 Chapter 3 49

 Fibrous proteins

Provide mechanical support

Often assembled into large cables or threads
-Keratins: major components of hair and nails
Collagen: major component of tendons, skin,
bones and teeth

Prentice Hall c2002 Chapter 3 50

Protein Purification Techniques

Purification steps usually exploit minor differences in the solubilities, net

charges, sizes, and binding specificities of proteins.

fractionation by ammonium sulphate

dialysis (gel filtration )
Column chromatography:
- Gel-filtration chromatography
- ion-exchange chromatography
- Affinity chromatography
Electrophoresis ( separaion by charge and mass)
polyacrylamide gel electrophoresis (PAGE), ( separaion by mass only)
2D Electrophoresis
HPLC, for high-performance liquid chromatography.
MS (Mass spectrometry)
GC-MS
MALDITOF (matrix-assisted desorption ionization- time-of-flight) MS

Prentice Hall c2002 Chapter 3 51

Amino Acid Composition of Proteins

Hydrolysis by 6 M HCl
Hydrolysis with phenylisothiocyanate (PITC) at pH 9.0

Prentice Hall c2002 Chapter 3 52

Column chromatography.

Prentice Hall c2002 Chapter 3 53

SDSPAGE

Prentice Hall c2002 Chapter 3 54

 Isoelectric Points
In acidic solution, the carboxylate and amine are in
their conjugate acid forms, an overall cation
In basic solution, the groups are in their base forms,
an overall anion
In neutral solution cation and anion forms are present
This pH where the overall charge is 0 is the
isoelectric point, pI

55
 Isoelectric Points
In acidic solution, the carboxylate and amine are in
their conjugate acid forms, an overall cation
In basic solution, the groups are in their base forms,
an overall anion
In neutral solution cation and anion forms are present
This pH where the overall charge is 0 is the
isoelectric point, pI

56
Prentice Hall c2002 Chapter 3 57
Prentice Hall c2002 Chapter 3 58
AMINO ACID METABOLISM
The Nitrogen Cycle and Nitrogen Fixation
Assimilation of Ammonia

A. Ammonia Is Incorporated into Glutamate and Glutamine

B. Transamination Reactions

Transfer of an amino group from an amino acid to an keto acid, catalyzed by a

transaminase. In biosynthetic reactions the initial amino acid is often In
transamination reactions involving glutamate as the amino group donor, the product is
ketoglutarate represents the precursor of a newly formed acid, (-amino acid)2.
Assimilation of ammonia into amino acids:

(a) The glutamate dehydrogenase pathway.

(b) Combined action of glutamine synthetase and glutamate synthase

under conditions of low NH4 concentration.
Synthesis of Amino Acids
The origins of the carbon skeletons of amino acids.
A. Aspartate and Asparagine
B. Lysine, Methionine, and Threonine
C. Alanine, Valine, Leucine, and Isoleucine
D. Glutamate, Glutamine, Arginine, and Proline

Conversion of glutamate to proline and arginine.

E. Serine, Glycine, and Cysteine

Biosynthesis of serine

Biosynthesis of glycine
Biosynthesis of cysteine from serine in many bacteria and plants.

Biosynthesis of cysteine in mammals

F. Phenylalanine, Tyrosine, and Tryptophan

Synthesis of shikimate and chorismate

Biosynthesis of phenylalanine and tyrosine from chorismate in E. coli.
The biosynthesis of tryptophan from chorismate requires five enzymes. In the
first step, the amide nitrogen of glutamine is transferred to chorismate; elimination
of the hydroxyl group and the adjacent pyruvate moiety of chorismate produces
the aromatic compound anthranilate . Anthranilate accepts a phosphoribosyl
moiety from PRPP. Rearrangement of the ribose, decarboxylation, and ring closure
generate indole glycerol phosphate.

Anthranilate.

The final two reactions of tryptophan biosynthesis are catalyzed by tryptophan

synthase:
 G. Histidine

Synthesis of histidine from phosphoribosyl pyrophosphate (PRPP) and ATP. Histidine is derived
from PRPP (5 C atoms), the purine ring of ATP (1 N and 1 C), glutamine (1 N), and
glutamate (1 N).
Amino Acids as Metabolic Precursors

A. Products Derived from Glutamate, Glutamine and Aspartate

Glu and Gln, are important players in nitrogen assimilation

Glu and Asp, are amino group donors in many transamination reactions and
required in the urea cycle.

Gln and Asp are also required as precursors in both purine and pyrimidine
biosynthesis.

Glu is required for synthesis of biologically active tetrahydrofolate.

B. Products Derived from Serine and Glycine
C. Synthesis of Nitric Oxide from Arginine

Conversion of arginine to nitric oxide and citrulline. NADPH is the source

of the five electrons.
Protein Turnover
Proteins are continually synthesized and degraded in all cells, a process
called turnover.

Their half-lives can vary from a few minutes to several weeks but the half-
life of a given protein in different organs and species is generally similar.

Protein Degradation 2 ways:

1. Lysosomes
2. Ubiquitin Proteasome System (UPS), ATP dependent
Overview of catabolism of amino groups & Excretory forms of nitrogen
 Degradation of Amino Acids

In animals, amino acids undergo oxidative degradation

in three different metabolic circumstances:

1. During the normal synthesis and degradation of cellular proteins (protein turnover),
some amino acids that are released from protein breakdown and are not needed for
new protein synthesis undergo oxidative degradation.

2. When a diet is rich in protein and the ingested amino acids exceed the bodys needs
for protein synthesis, the surplus is catabolized; amino acids cannot be stored.

3. During starvation or in uncontrolled diabetes mellitus,when carbohydrates are either

unavailable or not properly utilized, cellular proteins are used as fuel.
 Six Amino Acids Are Degraded to Pyruvate

Amino acids catabolized to

pyruvate are both ketogenic and
glucogenic. The six are:
alanine,
tryptophan,
cysteine,
serine,
glycine, and
threonine
Seven Amino Acids Are Degraded to Acetyl-CoA:
Five Amino Acids Are Converted to -Ketoglutarate
Four Amino Acids Are Converted to Succinyl-CoA
Asparagine and Aspartate Are Degraded to Oxaloacetate
 Isoelectric Points
In acidic solution, the carboxylate and amine are in
their conjugate acid forms, an overall cation
In basic solution, the groups are in their base forms,
an overall anion
In neutral solution cation and anion forms are present
This pH where the overall charge is 0 is the
isoelectric point, pI

89
 pI Depends on Side Chain
The 15 amino acids thiol, hydroxyl groups or
pure hydrocarbon side chains have pI = 5.0 to
6.5 (average of the pKas)
D and E have acidic side chains and a lower pI
H, R, K have basic side chains and higher pI

90
 Electrophoresis
Proteins have an overall pI that depends on the
net acidity/basicity of the side chains
The differences in pI can be used for separating
proteins on a solid phase permeated with liquid
Different amino acids migrate at different rates,
depending on their isoelectric points and on the
pH of the aqueous buffer

91
Amino Acid Catabolism
Amino acids obtained from the degradation of endogenous proteins or from the
diet can be used for the biosynthesis of new proteins.

Amino acids not needed for the synthesis of proteins are catabolized in order to
make use of their nitrogen and their carbon skeletons.

The first step in amino acid degradation is often removal of the group. Next, the
carbon chains are altered in specific ways for entry into the central pathways of
carbon metabolism.

Removal of the group of an amino acid occurs in several ways. The amino acid
usually undergoes transamination with --ketoglutarateto form an acid and
glutamate.

The glutamate is oxidized to and ammonia by the action of mitochondrial

glutamate dehydrogenase.

The net effect of these two reactions is the release of groups as ammonia and
the formation of NADH and -keto acids:
Overview of amino acid catabolism in mammals. The amino groups and
the carbon skeleton take separate but interconnected pathways.
Conversion of the carbon skeletons of amino acids to pyruvate, acetoacetate, acetyl
B. Arginine, Histidine, and Proline
C. Glycine and Serine
D. Threonine
E. The Branched-Chain Amino Acids
F. Methionine
G. Cysteine

The major route of cysteine catabolism is a three-step pathway leading to pyruvate.

Therefore, cysteine is glucogenic. Cysteine is first oxidized to cysteinesulfinate, which
loses its amino group by transamination to form -sulfinylpyruvat.
Nonenzymatic desulfurylation produces pyruvate
H. Phenylalanine, Tryptophan, and Tyrosine
I. Lysine
I. Lysine
The Urea Cycle Converts Ammonia into Urea
Discovered by Hans Krebs and Kurt Henseleit in 1932, (almost exclusively in liver).

In the first reaction, carbamoyl phosphate reacts in the mitochondrion with

ornithine to form citrulline in a reaction catalyzed by ornithine transcarbamoylase.

Citrulline is then transported out, in exchange for cytosolic ornithine.

The second nitrogen atom destined for urea comes from aspartate and is
incorporated when citrulline condenses with aspartate to form argininosuccinate.

Argininosuccinate is cleaved nonhydrolytically to form arginine plus fumarate

in an elimination reaction catalyzed by argininosuccinate lyase.

Final reaction of the urea cycle, the guanidinium group of arginine is hydrolytically
cleaved to form ornithine and urea in a reaction catalyzed by arginase.
The urea cycle.
 The exchange of glucose and alanine between muscle and liver, called the
glucosealanine cycle , provides an indirect means for muscle to eliminate nitrogen
and replenish its energy supply.

Glucosealanine cycle
Renal Glutamine Metabolism Produces Bicarbonate

The body often produces acids as metabolic end products; the resulting anions are
eliminated in the urine and the protons remain in the body.

Examples are: -hydroxybutyric axid, sulfuric acid.

These acid metabolites dissociate to give protons and the corresponding anion.

The blood has an effective buffer system for the protons, they react with bicarbonate.

to produce which is eliminated by the lungs (Fig). While this system effectively
neutralizes the excess hydrogen ions, it does so at the cost of depleting blood
bicarbonate. Bicarbonate is replenished by glutamine catabolism in the kidneys.

Glutamine -Ketoglutarate 2- + 2 NH4+

The overall process is:

2 Glutamin + 3 O2 + 6 H2O Glukosa + 4 HCO3- + 4 NH4+

 Nitrogen is fixed in only a few species of bacteria and blue-green algae by the
nitrogenase-catalyzed reduction of atmospheric to ammonia. Plants and
microorganisms can reduce nitrate and nitrite to ammonia.

Ammonia is assimilated into metabolites by the reductive amination of to glutamate,

catalyzed by glutamate dehydrogenase. Glutamine, a nitrogen donor in
many biosynthetic reactions, is formed from glutamate and ammonia by the action of
glutamine synthetase.

The amino group of glutamate can be transferred to an acid in a reversible

transamination reaction to form and the corresponding acid.

Pathways for the biosynthesis of the carbon skeletons of amino acids begin with simple
metabolic precursors such as pyruvate and citric acid cycle intermediates.

Nonessential amino acids are those that animals can produce in quantities that are
sufficient for growth. These amino acids are generally formed by short, energetically
inexpensive pathways. Essential amino acids must be supplied
in the diets of animals; these amino acids are synthesized by bacteria and plants.

In addition to their role in protein synthesis, amino acids serve as precursors in a

number of other metabolic pathways.
 Protein molecules in all living cells are continually synthesized and degraded.

Amino acids obtained from protein degradation or directly from food can be
catabolized. Catabolism begins with deamination, followed by modification of the
remaining carbon chains for entry into the central pathways of carbon metabolism.

The pathways for the degradation of amino acids lead to pyruvate, acetyl CoA, or
intermediates of the citric acid cycle. Amino acids that are degraded to citric acid
cycle intermediates are glucogenic. Those that form acetyl CoA are ketogenic.

In mammals, most nitrogen is excreted as urea, which is formed by the urea cycle in
the liver. The carbon atom of urea is derived from bicarbonate. One amino group is
derived from ammonia, and the other from aspartate.

The metabolism of glutamine in the kidneys produces the bicarbonate needed to

neutralize acids produced in the body.