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Biochemistry Exam 2 LGT

Heinz Schwarzkopf

hs0215@live.unthsc.edu

LGT Disclaimer:
LGTs are designed by CAP tutors. They are not meant to replace information you
receive in class or textbooks. While it has been reviewed for typos and errors, you
must always remember that your professor and the material given by them
supersede any information presented in the LGT. The LGT serves as a review, and
are not meant to be an all-inclusive presentation.

Bioenergetics and Metabolism


Glycolysis
PDH & TCA Cycle
Electron Transport System
Oxidative Phosphorylation

Metabolic pathways are divided into two categories:


Anabolic pathway: Requires energy
gluconeogenesis.

Catabolic pathway:
glycolysis

What type of metabolic pathway is


the TCA cycle?

Catabolic

Anabolic

Pathways in Carbohydrate Metabolism

What stops these two pathways from occurring at the same


time?

Metabolic Pathways are:


Compartmentalized.
Driven by the net free energy change of

the sum of individual reactions.


Regulated.

RELEVANT CELLULAR COMPARTMENTS


Cytosol / cytoplasm
Glycolysis, portions of gluconeogenesis, glycogen

metabolism, pentose phosphate pathway.


Mitochondria
TCA, electron transport, >90% of ATP synthesis,

fatty acid oxidation.

Regulation

Allosteric effectors (e.g., ATP) VERY FAST!!


Some metabolic enzymes are regulated by allosteric effectors. An
allosteric effector may be a substrate, intermediate or product of the
pathway.

Short-term (at the protein level): some enzymes are


modified covalently by phosphorylation. This regulatory mechanism
requires a protein kinase (phosphorylating enzyme) and protein
phosphatase (dephosphorylating enzyme).
Covalent modification (e.g. phosphorylation) FAST!....

Long-term (at the DNA level): the amount of the enzyme/protein


is adjusted by a change in its synthesis or degradation.

Change enzyme amounts - SLOW. Metabolic enzymes have life


spans ranging from 1 hour to several days; therefore this is a long-term
regulation.

Feedback Inhibition and Feedforward Stimulation


Many reaction products are used as the reaction feedback inhibitors
ATP is used as feedback inhibitor in many catabolic processes
Substrate A

Product E

In feedforward stimulation, a
substrate stimulates the pathway by
which it is utilized.

What class of enzyme is a kinase?

Inherited Enzyme Deficiencies cause


Metabolic Diseases
When an enzyme is missing as a result of a genetic
defect, the immediate effects are always the
accumulation of the substrate and the lack of the
product.
If the intracellular degradation of a macromolecule (lipid,
polysaccharide, etc.) by hydrolytic enzymes is blocked,
the undegraded molecule accumulates mostly within the
cells. The result is a "storage disease.

Oxidation-reduction: reactions involve the loss or gain


of electrons (or protons) one reactant gains electrons
and is reduced while the other loses electrons and is
oxidized.
Oxidation reactions generally release energy and are
important in catabolism. (beta Oxidation is an oxidation or
reduction taking place?)
What is the fate of glucose?

6CO2 + 6H2O
When you see Dehydrogenase that is a clue that you are
doing a redox reaction. Then remember that an
NADH/NADPH/FADH2 is either used or produced.

NAD and FAD


1.

In some enzymes bound covalently, called prosthetic


group, e.g. succinate dehydrogenase.

2.

NAD+ or NADP+ are a soluble co-factor free to diffuse


from one enzyme to another. Both accept two electrons
and one proton. Derived from vitamin B3 niacin
FAD or FMN are coenzyme derived from the vitamin
riboflavin, B2, often tightly bound to specific enzymes
called flavoproteins.

3.

4.

Great majority of NAD+/NADH is located in the


mitochondria.

5.

Most of NADP+/NADPH is in the cytosol.

Electron Carriers
Both coenzymes (NAD+ and NADP+) undergo reversible reduction of
the nicotinamide ring.
What can niacin deficiency lead to?
a) Rickets
b) Diarrhea
c) Sterility
d) Dermal sensitivity

Pellagra: Remember Niacin and the four Ds

Rossman fold What it for?


Most dehydrogenases that use NAD or NADP bind the
cofactor in a conserved protein domain, called Rossmann
fold.

FAD/FADH2
Flavoproteins are enzymes that catalyze oxidation-reduction
reactions using either FMN or FAD as coenzyme. FAD or
FMN are coenzyme derived from the vitamin B2, riboflavin.

Flavin Adenine Dinucleotide

Adenine
N-glycosidic bond

In a Healthy Cell
NAD+

NADP+

NADH

NADPH

What is NADPH used for?

Metabolism
All major nutrients (carbohydrates, fat, protein, etc.)
are degraded to acetyl coenzyme A (acetyl-CoA)
Think of acetyl-CoA as the center molecule of metabolism

In the mitochondria, the two carbons of the acetyl


group become oxidized to CO2 in the TCA cycle, but
may be used to produce all your other molecules.

Do you remember what vitamins B2 and 3 are for?

Which of the following ways can the


body not obtain glucose?
A.
B.
C.
D.
E.

Monosaccharides
From the breakdown of glycogen
Glucose synthesis via amino acids
Glucose synthesis via acetyl CoA
Glucose can be obtained from all of these
sources

Sources and Fates of Acetyl-CoA

Glucose: Sources, Uptake, Transport, Storage and


Oxidation
Glucose occupies a central position in the metabolism of plants, animals and
microorganisms.
Glucose is the principal transported carbohydrate in humans and most
abundant monosaccharide in dietary carbohydrates.
Glucose can be transported by the blood, but it cannot be stored in the cells.
For storage, it has to be converted to the polysaccharide glycogen.
What are the organs that store glycogen?

Which of the following glucose


transporters is insulin dependent?
A.
B.
C.
D.
E.

Glut1
Glut2
Glut3
Glut4
Glut5

What about the kidneys?


If a person is on a salt diet and has not
ingested salt in 3 days can there intestines
still absorb glucose?

Why are certain organs such as the


brain and retinas affected by high
blood glucose levels?
a) Because they run out of glucose transporters
b) Because they dont have a sweet tooth
c) Because glucose freely diffuses into the cells and is
toxic above certain levels
d) Because they cant use insulin

21

During the well-fed state: decreased levels of glucagon


and elevated levels of insulin
During starvation: elevated levels of glucagon and low
levels of insulin

Glucose metabolism by the liver depends on the


insulin/glucagon but insulin is NOT required for the entry
of glucose into the liver cell. Why?

What about entry into pancreatic islet cells?

Why doesnt the muscular glycogen store


contribute to gluconeogenesis?
a) Muscles are stingy
b) Muscles use up all the glucose before it can leave
c) Because it cant convert glucose-1-phosphate to glucose-6phosphate

d) Because it lacks glucose-6-phosphatase

23

Which of the following is only depend


on glucose for energy?
A.
B.
C.
D.
E.

Brain
Red Blood Cells
Liver
Adipose Tissue
Skeletal Muscle

Remember blood glucose maintained at 5 mmol/liter

Which of the following enzymes is involved in an irreversible


step in glycolysis?
A. Hexokinase
B. Phosphoglycerate kinase
C. Glyceraldehyde-3-phosphate DH
D. Enolase
E. Phosphoglycerate mutase
What does the enzyme do?

How many irreversible steps?


Where does glycolysis take place?

Glycolysis: the reactions of


glycolysis, the major catabolic
pathway for glucose. It is active in
the cytoplasm of all cells in the
human body.

6 - Carbon sugar
first priming reaction
Step 1

Breakdown of the six-carbon


glucose into two molecules of
three-carbon pyruvate, occurs in
10 steps and 2 phases:
Step 2

1. energy investment phase:


(steps 1 5)
Phosphorylation of glucose
and its conversion to
glyceraldehyde 3-phosphate

second priming reaction


Step 3

Step 4

3 - Carbon sugar

3 - Carbon sugar

Step 5

2. energy generation phase:


(steps 6 10)
Oxidative conversion of
2
glyceraldehyde 32
phosphate to pyruvate

2
Step 6

Oxidation and phosphorylation

2
Step 7

first ATP forming reaction

2
Step 8

Be able to walk yourself


Through the steps.

2
Step 9

2
2

No oxygen needed

Second ATP forming reaction


Step 10

Which of the following enzymes is only found in the liver


and pancreas?
A. Hexokinase
B. Phosphoglycerate kinase
C. Glucokinase
D. Aldolase
E. Pyruvate kinase
Remember that a kinase catalyzes and irreversible
reaction.

Phosphorylation of glucose traps it in the cell (neg charge).

In the liver, the enzyme that converts glucose to glucose-6phosphate is:


A. Inhibited by ATP
B. Inhibited by ADP
C. Inhibited by Glucose 6-phosphate
D. Inhibited by Insulin
E. Inhibited by Fructose-6-phosphate
E. In peripheral tissue, hexokinase is inhibited by its product,
Glucose-6-P, but glucose-6-P has no effect on glucokinase (which
is found in the liver and pancreas)

Glucokinase (Liver & Pancreas)


- Highly specific for glucose.
- High Km for glucose (~10 mM).
- Not saturated at physiological blood glucose concentrations
(4 - 5 mM).

- Inhibited by fructose 6-P, but NOT glucose 6-phosphate.


- Induced by insulin (transcriptionally).

The liver acts like a glucose sponge

Hexokinase (Peripheral Tissues)


- Low specificity - phosphorylates most relevant hexoses
(glucose, fructose, mannose).
- Low Km (0.1 mM); saturated at all plasma glucose
concentrations.

- Inhibited by glucose 6-phosphate.


- Activated by fructose 1-phosphate and glucose.

- Insulin has no effect on expression.

Km of the two isozymes of hexokinase

50

What are the products of gylcolysis?

Cytosolic NADH+
- It must be re-oxidized:
1. anaerobic conditions: by conversion of pyruvate to lactate by
lactate dehydrogenase, and NADH is re-converted to NAD+,
without oxygen participation.

An increase in intracellular ATP in working muscle would


cause?
A. an decrease in glycolysis
B. an decrease in glycogen synthesis
C. An decrease in gluconeogenesis.
D. Activation of pyruvate kinase
E. Activation of PFK-1

Regulation of Glycolysis
- Phosphofructokinase-1 (PFK-1), is the most important regulated
enzyme of glycolysis. PFK is:
- stimulated by insulin and inhibited by glucagon (in the liver).
- inhibited by citrate as well as by low pH.
- inhibited by ATP and stimulated by AMP and ADP.
- Hexokinase / Glucokinase: also regulated
- Pyruvate Kinase: also regulated. Inhibited by ATP.

- In RBCs, rates of glycolysis are regulated by ATP / (AMP+ADP) only.

Remember 1, 3 and 10 those are the


irreversible steps of glycolysis.

Phosphofructokinase-1(PFK-1)
- Is the most important regulated enzyme of glycolysis.
- Commits glucose to pyruvate in glycolysis.
- Activated by:
- AMP
- Fructose 2,6-bisphosphate synthesized by PFK-2
- levels determined by insulin/glucagon
- levels determined by fructose 6-phosphate in muscle
- Inhibited by:
- ATP
- Citrate
- Glucagon

2,3-bisphosphoglycerate in Red blood cells.

2,3-bisphosphoglycerate is an important allosteric effector of


hemoglobins affinity for oxygen.
It shifts the oxygen binding curve to right = DECREASED affinity,
resulting in more efficient release of oxygen at tissues.

Pyruvate Kinase
In glycolysis, step 10, phosphoenolpyruvate is converted to
pyruvate by pyruvate kinase. Pyruvate kinase is more
active in the fed state than in the fasting state.
- inhibited by:

- ATP, acetyl-CoA, alanine


- activated by:
- Fructose 1,6-bisphosphate

Fates of Pyruvate
Pyruvate formed by glycolysis, is only the first stage in the complete
degradation of glucose.
First route: pyruvate is oxidized, with loss of its carboxyl group as
CO2, to yield acetyl group of acetyl-CoA by PDH. Acetyl group is
then oxidized to CO2 by TCA cycle.
Second route: pyruvate is reduced to lactate by lactate
dehydrogenase via lactic acid fermentation.
Third route: pyruvate catabolism leads to alcohol and CO2, under
hypoxic conditions, a process called alcohol fermentation.

What is the fate of glucose:


a) Will be used up faster under anaerobic
condition than aerobic conditions
b) Will be used up faster under aerobic conditions
than anaerobic conditions
c) Will produce 36-38 ATP in all cells
d) Will be used to produce 2,3-BPG and this is
know as the Warburg effect
What is this phenomenon called and where is it not
seen?
What is the Warburg effect?

[1] Lactate Is Produced under


Anaerobic Conditions

Fructose:
a) is highly regulated
b) primarily metabolized by muscle
c) Uses GLUT5 transporter
d) is essential in fructosuria patients

42

FRUCTOSE ENTRY INTO LIVER


METABOLISM
glucokinase

Disorders of Fructose Metabolism


1

Essential fructosuria: a deficiency of fructokinase.


-asymptomatic

Hereditary fructose intolerance: a deficiency of fructose


1-phosphate.
-accumulation in your cells

Pryuvate is formed in the ______ and oxidized in


the ______.
A. cytoplasm
B. mitochondrial matrix
C. cytoplasm
D. mitochondrial inner space
E. cytoplasm

Cytoplasm
cytoplasm
mitochondrial membrane
mitochondrial matrix
mitochondrial matrix

Which of the following is a cofactor for pyruvate


dehydrogenase complex (PDC)?
A. NADP+
B. ATP
C. Vitamin B1
D. FADH2

What is Thiamin used for?

Glycolysis
PDH
TCA
ETC

Pyruvate Dehydrogenase Complex (PDC)


- Pyruvate, derived from glucose and other sugars by glycolysis, is
oxidized to acetyl-CoA and CO2 by PDH complex.
The three enzymes that make up the complex:
E1 pyruvate dehydrogenase; It is inhibited by its products NADH
and acetyl-CoA and stimulated by AMP. It is inactivated by the
phosphorylation and reactivated by dephosphorylation.
E2 dihydrolipoyl transacetylase, catalyzes the transfer of the acetyl
group to coenzyme A, forming acetyl-CoA.
E3 dihydrolipoyl dehydrogenase, catalyzes the regeneration of the
disulfide (oxidized) form of lipoate.
regulatory enzymes: phosphorylate / dephosphorylate E1 (a kinase
and phosphatase). The kinase is activated by ATP, thus ATP
inhibits E1.

The overall reaction is irreversible; fat cannot be converted into


carbohydrate.

Why cant fat be converted into glucose?


a) As a means to punish people who cant cook
b) Because anaperotic reactions are inhibited during
the fasting state
c) Because the PDH reaction is irreversible
d) Because of an accumulation of oxaloacetate

Key for the future:


Phosphorylation of gluco-metabolic
enzymes tends to correlate with getting
energy out.
Dephosphorylation of gluco-metabolic
enzymes tends to correlate with
producing ATP.

Mechanism of PDH Action

Five consecutive reactions:


TPP lipoic acid CoA FAD NAD
Decarboxylated then transferred to CoA

Pyruvate Dehydrogenase Complex (PDH)


Requires 5 different cofactors:

Thiamine pyrophosphate, TPP (B1) - E1


Lipoic acid (lipoate) - E2
Flavin adenine dinucleotide, FAD (Riboflavin) - E3
Nicotinamide adenine dinucleotide, NAD+ (Niacin)
Coenzyme A (CoA) (synthesized from pantothenic
acid (B5)

Make sure youve read through the details on the PP.

Thiamine Pyrophosphate (TPP)


- removal of carboxyl (-COOH) groups from organic acids
- is a vitamin B1 derivative which is produced by the enzyme thiamine

Thiamine deficiency leads to inability to oxidize pyruvate and thus


has a major neurological impact (Beriberi).

Regulation of PDH
Allosteric
- Inhibition by ATP, acetyl-CoA and NADH.

- Activation by AMP, CoA and NAD+.


Covalent
- Activation by dephosphorylation
- Inhibition by phosphorylation of E1 (pyruvate
dehydrogenase). Kinase activated by ATP.
Pyruvate is a potent inhibitor of pyruvate
dehydrogenase kinase.

TCA Cycle
Degradation of acetyl-CoA derived from carbohydrates, fatty acids
and amino acids.
- produces most of the CO2 generated in tissues (2).
- is the major source of NADH (3).
- allows excess energy to be used for fatty acid biosynthesis.
- provides precursors for many metabolites.
Most TCA cycle enzymes in the mitochondrial matrix; some in the

inner mitochondrial membrane.

How many molecules of NADH are formed from the


breakdown of 1 molecule of pyruvate during one
complete turn of the TCA cycle?
A.
B.
C.
D.
E.

1
2
3
4
5

Remember: 2 in glycolysis, and one more than


that in TCA

TCA Strategy
- Activated 2C fragment (acetyl-CoA) reacts with a 4C oxaloacetic acid, to
yield 6C citrate.
- In a series of seven reactions two carbons are released as CO2 (oxidative
decarboxylations), regenerating oxaloacetate.
- 2 carbons in, 2 carbons out = no net gain.
- Oxaloacetate must be present to "prime" the cycle.
- Four pairs of electrons are transferred during one turn of the cycle: three
pairs of electrons reducing three NAD+ to NADH, and one pair reducing
FAD to FADH2.

- One complete turn of the TCA cycle generates only one ATP (GTP) (in the
conversion of succinyl-CoA to succinate), three molecules of NADH and
one molecule of FADH2. Oxidation of one NADH leads to formation of
3 ATP, whereas oxidation of FADH2 yields 2 ATP.
-

Total 12 ATP generated by one round of TCA cycle from one acetyl residue. From
two acetyl residues, total 24 ATP generated.

Know the 8 steps

TCA Cycle

Acetyl-CoA

citrate
synthase

2
malate
dehydrogenase

aconitase

TCA Cycle
isocitrate
dehydrogenase

fumarase

hydration

succinate
-ketoglutarate
dehydrogenase dehydrogenase
succinyl-CoA
synthetase

(also called oxoglutarate)

6
5

(ATP)

phosphorylation

4
oxidative decarboxylation

Which of the following is an example of substrate


level phosyhorylation:
A. Fumarase.
B. Alpha-Ketogluterate Dehydrogenase
C. Hexokinase
D. Succinyl-CoA Synthetase.
E. PFK1
Synthetase vs. Synthase
What is the total ATP yield from glycolysis plus TCA?
-but what about ETC?

Regulation of the
TCA Cycle
Regulation of metabolite flow
from the PDH complex through
the TCA cycle in mammals.
The PDH complex is allosterically
inhibited when [ATP]/[ADP],
[NADH]/[NAD+], and [acetylCoA]/[CoA] ratios are high,
indicating an energy-sufficient
metabolic state. When these ratios
decrease, allosteric activation of
pyruvate oxidation results.

The overall rate of the TCA cycle is controlled


by the rate of conversion of pyruvate to acetyl-CoA.

Anaplerotic Pathways
TCA cycle intermediates
Pyruvate carboxylase, malic enzyme, PEP-carboxykinase. Four rxns

Pyruvate Carboxylase Defficiency


A rare recessively inherited condition (enzyme
deficiencies are normally genetic recessive)
Pyruvate accumulates because pyruvate carboxylase is a
major consumer of pyruvate
Accumulated pyruvate is converted to lactate and alanine
Hypoglycemia
Caused by the inability to convert pyruvate to glucose

Neurological deficits and mental retardation


Dysfunction of TCA cycle due to insufficient oxaloacetate
Energy dysfunction normally results with neurological problems

Antiporters Transport Metabolites Across the


Inner Mitochondrial Membrane
Transport across the inner mitochondrial
membrane usually requires specific carriers
ADP, ATP (but not the other nucleotides);
Pyruvate; Phosphate; Alanine, Aspartate;
Glutamate; Substrates, intermediates and
products of the TCA cycle
except: acetyl-CoA, oxaloacetate, fumarate, NAD,
NADH (3ATP)
Does NADH really not need a transporter?

UNDER AEROBIC CONDITIONS (1)

Regeneration of NAD

What about the outer


membrane?

UNDER AEROBIC CONDITIONS (2)


Skeletal and cardiac muscle

More efficient 1 to 1 transfer

Electron transport chain (respiratory chain)


and oxidative phosphorylation
- Reaction intermediates donate electrons to specific coenzymes, such
as NAD+ and FAD to form the energy-rich reduced coenzymes, NADH
and FADH2.
- These reduced coenzymes can donate a pair of electrons to a electron
carriers, collectively called electron transport chain, and they lose their
free energy, that can be stored by the production of ATP from ADP and Pi,
this process is called oxidative phosphorylation.
- The electron transport chain is present in the inner mitochondrial
membrane.
- Electron transport chain and oxidative phosphorylation (ATP synthesis): are
tightly coupled processes. Therefore, inhibition of the electron transport
chain also results in inhibition of ATP synthesis.

Oxidative phosphorylation
Oxidative phosphorylation involves the reduction of O2 to H2O with
electrons donated by NADH and FADH2.
Photo-phosphorylation involves the oxidation of H2O to O2, with
NADP+ as electron acceptor.
Oxidative phosphorylation and photo-phosphorylation, both are
mechanistically similar both processes involve the flow of electrons.
Oxidative phosphorylation begins with the entry of electrons into the
respiratory chain.
NADH (carries electrons from catabolic reactions) and NADPH (supplies
electrons to anabolic reactions), are water-soluble electron carriers that
associate reversibly with dehydrogenases.

Major Mitochondrial Electron Carriers


(or Electron-Transfer System or Respiratory Chains)
None of the functional groups in proteins can transfer hydrogen or
electrons, the components of the respiratory chain have to employ
metal ions and coenzymes.
Respiratory chains contains: (e- carriers)
-

Flavoproteins
Iron-sulfur proteins
Cytochromes
Ubiquinone (UQ) or Coenzyme Q (CoQ)
Protein-bound copper

Note: With the exception of coenzyme Q, all members of this chain


are proteins.

69

Flavoproteins
- The FAD or flavin mononucleotide (FMN) in the flavoproteins can
transfer either one- or two-electrons at a time.
- Electron transfer occurs because the flavoprotein has a higher
reduction potential than the compound oxidized.
- They accept hydrogen/electrons from NADH and donate it to the
cytochromes.

Prosthetic group (tightly bound) to complex 2

Iron - Sulfur Proteins


(e- carriers)

- also known as non-heme iron proteins. What is a heme?


- anchored in proteins through cysteine.
- both the iron-sulfur proteins and the flavoproteins of the respiratory
chain are integral membrane proteins.

Fe3+ + e- Fe2+

Iron-sulfur complexes in proteins: iron in these complexes can change its


oxidation state reversibly between the ferrous (Fe2+) and ferric (Fe3+) forms.

Cytochromes
Cytochromes: Heme proteins (is a complex of protoporphyrin IX and
ferrous iron) that functions as electron carriers in respiration,
photosynthesis and other oxidation-reduction reactions.
- One-electron carriers (Fe3+ Fe2+).
- All but one (cytochrome c) are integral membrane proteins.
- Cytochrome c is a small and water-soluble protein.
What does it do?

Ubiquinone (UQ) or Coenzyme Q (CoQ)


- Ubiquinone, a hydrophobic quinone, is a lipid-soluble benzo-quinone
with a long isoprenoid side chain.

Makes molecule hydrophobic

Match the following


with where they are
found:

A. Fe-S proteins
B. Flavin proteins
C. Cytochrome C
D.Cytrochome b
E. Coenzyme Q
(ubiquinone)

1. Integral
membrane
protein
2. Free to diffuse

Which of the following


complexes contain a proteinbound copper?
A.NADH dehydrogenase
B.Succinate Dehydrogenase
C.Cytochrome b-c Complex
D.Cytochrome c Oxidase
What are each of the above
mentioned structures?

Protein-bound copper
- Protein-bound copper participates in the last reaction of
the respiratory chain, the transfer of electrons to molecular
oxygen. It switches between the Cu+ and Cu2+ forms during
these electron transfers. Part of complex 4

Respiratory Chain
Four members of the respiratory chain are freely diffusible:
NADH, ubiquinone, cytochrome c, and molecular oxygen.
1 Complexes I and II catalyze electron transfer to ubiquinone from two
different electron donors: NADH (complex I) and succinate or
FADH2 (complex II).
2 Complex III carries electrons from reduced ubiquinone to cytochrome
c, and
3 Complex IV transferring electrons from cytochrome c to O2.

Which side of the mitochondrion


is the most acidic after oxidative
phosphorylation?
A.The inner membrane space
B.The matrix
C.Outer membrane space
D.pH is the same across
Cells with high rates of respiration (e.g. heart
muscle) have mitochondria with many
densely packed cristae. Liver cells have
much fewer cristae.

COMPONENTS OF THE
ELECTRON TRANSPORT CHAIN
COMPLEX I

COMPLEX III

COMPLEX IV

Increasing reduction potential

What happened to the last 2H+ at complex 4?

Complex I

(NADH to Ubiquinone)
- Complex I, also called NADH:ubiquinone oxidoreductase or
NADH
dehydrogenase, transfers electrons from NADH to ubiquinone, is a
large enzyme composed of 42 different polypeptide chains, including
FMN-containing flavoprotein and at least six iron-sulfur centers.
-no FAD?
- Electrons are transferred from NADH -> FMN -> ironsulfur -> ubiquinone.

- Amytal (a barbiturate drug), rotenone (a plant product commonly


used as an insecticide), and piericidin A (an antibiotic), inhibit electron
flow from the Fe-S centers of Complex I to ubiquinone.
If you block complex 1 can oxidative phosphorylation take place?

Complex II
(Succinate to Ubiquinone)
Complex II, also called succinate dehydrogenase, is the only
membrane-bound enzyme in the TCA cycle.

- It contain three Fe-S centers, bound FAD


- Electrons from succinate pass through a flavoprotein and several Fe-S
centers to ubiquinone.
- Acyl-CoA dehydrogenase (the first enzyme of oxidation) transfers electrons to ETF
(electron-transferring flavoprotein, FAD), from which they pass to ubiquinone.

Complex III
(Ubiquinone to Cytochrome c)
Complex III, also called cytochrome c oxidoreductase or cytochrome
bc1 complex, couples the transfer of electrons from ubiquinol (QH2) to
cytochrome c.
- It contains cytochrome b, an Fe-S protein, and cytochrome c1.
- Cytochrome c is a soluble protein. After its single heme accepts an
electron from Complex III, cytochrome c moves to Complex IV to
donate electron to a binuclear copper center.

INHIBITED BY ANTIMYCIN A

Complex IV
(Cytochrome c to O2)
This is the final step of the respiratory chain. Complex IV, also called
cytochrome oxidase, carries electrons from cytochrome c to molecular
oxygen, reducing it to H2O.
- Complex IV is a large enzyme (13 subunits; Mr 204,000) of the inner
mitochondrial membrane.
- Oxygen is tightly bound between heme a3 and copper, to be released
after its reduction to H2O by the transfer of four electrons.

- Cytochrome oxidase has a very high affinity for oxygen.

- INHIBITED BY: CYANIDE, CO, AZIDE

ATP Synthase
O for Oligomycin

F1

FO

84

Proton Gradient
Oxidative phosphorylation proceeds in two steps:
1. Protons are pumped out of the mitochondrion: Proton pumping is
driven by the redox reactions in the respiratory chain, and creates a
electrochemical gradient across the inner mitochondrial membrane.
The inner membrane must be impermeable to protons to maintain
gradient.
Protons are admitted back into the mitochondrion, down their
concentration gradient, via proton channel: This process drives
ATP synthesis.
- four protons are pumped by the NADH-ubiquinone reductase complex
and another four by the ubiquinone-cytochrome c reductase complex.
- cytochrome oxidase removes four protons from the mitochondrial
matrix, translocating two to the inter-membranous space and other two
consuming in the reduction of O2, thus, four protons leads to the synthesis
of one ATP molecule.
- ATP is synthesized only when protons flow, and protons can flow

only when ATP is synthesized.

Respiratory Control
-

There is no rate-limiting step in oxidative phosphorylation, but its


rate depends on substrates availability (ADP, inorganic phosphate
and O2), and an oxidizable metabolite, like NADH and/or FADH2.

ATP synthesis is absolutely dependent on continuous electron flow


(electron transport) and electron transport occurs only during ATP
synthesis.

Oxidative phosphorylation produces most of the ATP.

In healthy cells, the level of ATP exceeds that of ADP by a factor of


4 - 10.

High demand for ATP, generates ADP, which stimulates respiration.

At rest, accumulation of ATP depletes ADP levels and suppresses


respiration.

2,4-dinitrophenol:
A. Inhibits the flow of electrons at complex 4
B. lowers the basal metabolic rate
C. Inhibit the flow of electron at complex 2
D. decrease the H+ gradient across the inner
mitochondrial membrane

Uncouplers do which of the following:

A. Diminish the proton gradient


B. Disrupts the electron transport chain
C. Prevent Complex I from transferring electrons from
NADH to ubiquinone
D. Disrupts oxidative phosphorylation but allows ATP
synthesis to occur

Uncouplers of Oxidative Phosphorylation


Uncoupler - Any substance that inhibits ATP synthesis but have no effect on
electron transport.
(Uncouplers means the dissociation of oxidative phosphorylation (not
reduction) from ATP synthesis. The most common uncoupler is 2,4dinitrophenpl (DNP)).
specific chemical agents:

- 2,4-dinitrophenol (DNP) and pentachlorophenol


- Valinomycin: an antibiotic that makes the inner mitochondrial
membrane permeable for potassium.
Oxygen utilization is maintained as electron transport (and H+ pumping)
continue.
ATP synthesis is diminished or inhibited, as proton gradient (electronflow) is weak or disrupt, or non-existent.

Uncouplers of Oxidative Phosphorylation


physiological un-couplers:
- Thyroxin: a hormone produced by the thyroid glands to regulate
metabolism by controlling the rate of oxidation in cells.
- Thermogenin: a protein of the inner mitochondrial membrane that
allows trans-membrane movement of protons, also called uncoupling
protein, a membrane-spanning protein that forms a natural
channel to allow proton return.
increases heat generation.
found in specialized tissues (usually hairless, hibernators or coldadapted).
brown fat (particularly high content of cytochromes) localized in
the neck and upper back.

Inhibitors of Electron Transport


Oxidative phosphorylation is inhibited by many poisons.
Electron flow through the respiratory chain, can be blocked by:
Rotenone - an insecticide, blocks flow of electrons from Fe-S complexes in the
NADH-Q reductase complex to ubiquinone through Complex I,
(Parkinsons?). Blocks transfer of electrons associated with NADH.
Amytal (a barbiturate), site of action same as rotenone, also inhibit electronflow through the NADH-Q reductase complex (Complex I).
Antimycin A, an antibiotic, that blocks electron flow from cytochrome b to c1 through cytochrome c oxido-reductase complex (Complex III).
Cyanide - binds to cytochrome oxidase (Complex IV) and prevents electron
transfer to oxygen. Hydrogen sulfide, carbon monoxide and azide also act
as cyanide.

Mitochondrial DNA
Mutations in mitochondrial DNA can cause:
- Lebers hereditary optic neuropathy (LHON): blindness, caused by degeneration
of the optic nerve.
- Myoclonic epilepsy and ragged-red fiber disease (MERRF).
- Mitochondrial DNA has a higher mutation rate than nuclear DNA.

92

Which of the following can be damaged by


ROS?
A. Hemoglobin
B. Proteins
C. DNA
D. Cell Membranes
E. All of the above

Reactive Oxygen Species (ROS) Are Formed


During Oxidative Metabolism
Electrons may leak out of the respiratory chain:
Superoxide: O2 + e O2
H+

O2

HO2

HO2

O2

H2O2

H+

o Also: Reduced flavoproteins (FADH2) in locations other


than the respiratory chain can produce hydrogen
peroxide (H2O2)

Hydroxyl radical:
H2O2 + Fe2+ + H+ HO + Fe3+ + H2O

Enzymes Evolved to Destroy ROS


Superoxide dismutase (SOD)
2O2 + 2H+

SOD

H2O2 + O2

Catalase (a heme-containing enzyme that


destroys H2O2)
2H2O2

Catalase

2H2O + O2

Peroxidases
Glutathione peroxidase (2GSH + H2O2 GSSG + 2H2O)

Many Metabolites, Vitamins, Hormones and


Phytochemicals Can Eliminate Dangerous Free
Radicals
These molecules form stable free radicals that are
sufficiently reactive to react readily with free radicals
but not sufficiently reactive to damage normal
constituents of the cell.

Bilirubin
Uric acid
Ascorbate (Vitamin C)
Estrogens

Carbohydrate Metabolism
Most important function of carbohydrate metabolism is: maintain blood
glucose level at all times.
- Brain alone consumes ~ 120 g of glucose / day. Therefore, a blood glucose
level of 4.0 to 5.5 mmol/liter (70 to 100 mg/dL) must be maintained at all
times.
-

Only liver glycogen (NOT muscle glycogen), can be used to


maintain the blood glucose level.

- Gluconeogenesis produces glucose from amino acids, lactic acid, and


glycerol. It is the only source of glucose during prolonged fasting.
- Only the liver and kidneys have a complete gluconeogenic pathway.
-

Liver produces 10 times more glucose than kidney, because the liver is so
much larger than a kidney.

Which of the following cannot be utilized for the


net synthesis of glucose?
A. Leucine
B. Glycerol
C. fatty acids
D. Aspartate
E. Alanine
F. Acetyl CoA

Remember that the 2 L amino


acids are only ketogenic.

Gluconeogenesis new formation of sugar


Glucose stored in the liver and muscle, in the form of glycogen. But this stored glucose
is not sufficient between meals and fasting. At that time, organisms synthesize glucose
from non-carbohydrate (such as pyruvate). This pathway called gluconeogenesis.

- Occurs primarily in the cytosol, although some precursors are


generated in the mitochondria.
- Glucose is a universal fuel and building block in humans, some tissues
(brain, erythrocytes, testes) depend almost completely on glucose
for their metabolic energy.

- Brain alone requires ~ 120 g of glucose each day this is more than
half of all the glucose stored as glycogen in liver and muscle.
- Liver is the major gluconeogenic organ of the body.
- Muscle does NOT contribute to gluconeogenesis, because

no glucose 6-phosphatase and no glucagon receptors

Gluconeogenesis
Gluconeogenesis: synthesis of carbohydrate (such as glucose)
from non-carbohydrates such as oxaloacetate or pyruvate
(Synthesis of one molecule of glucose from two molecules of pyruvate
requires energy, equivalent to 6 molecules of ATP)

- An energy-requiring, biosynthetic pathway of generating


glucose, generally from 3- and 4- carbon compounds.
- Gluconeogenesis must get around the highly exergonic
glucokinase, phosphofructokinase-1 (PFK-1) and pyruvate
kinase reactions by a new set of exergonic reactions that
drive the process in the opposite direction. (1, 3 & 10)

Gluconeogenesis
Gluconeogenesis and glycolysis are NOT identical pathways,
running in opposite directions. Reverse of glycolysis except
the irreversible (kinase) reactions.
Three reactions of glycolysis are irreversible and cannot be used in
gluconeogenesis:

1. Conversion of glucose to glucose 6-phosphate by


hexokinase/glucokinase.
2. Phosphorylation of fructose 6-phosphate to fructose 1,6bisphosphate by phosphofructokinase-1 (PFK-1).
3. Conversion of phosphoenolpyruvate to pyruvate by pyruvate
kinase.

Match the glycolytic enzymes with their gluconeogenic


counterparts:

1. Hexokinase/
Glucokinase
2. PFK-1
3. Pyruvate Kinase

A. PEP carboxykinase
B. Fructose -1,6bisphosphatase
C. Pyruvate carboxylase
D. Glucose-6phosphatase

Glycolysis

Gluconeogenesis
[10]

[8]

Glycolysis and
Gluconeogenesis
Irreversible reactions # [1], [8],
and [10] Know the enzymes.

7
6
5
4
3
2
[1]
Notice the use of an ATP and a GTP

Which of the following liver enzymes would be active


in the fasted state?
A. pyruvate kinase
B. PEP carboxykinase
C. Fructose 1,6 bisphosphatase.
D. Both A and B
E.Both B and C

Which of the following metabolic reactions can


pyruvate carboxylase be found?
A. Glycolysis
B. Krebs cycle
C. Glycogen synthesis
D. Gluconeogenesis
E. Both B and D
F. Both A and D

Conversion of Glucose 6-phosphate to Glucose

- The irreversible glucokinase reaction is reversed by the


action of glucose-6 phosphatase (a simple hydrolysis). This
enzyme is located in the ER and is present only in

the liver.
- Glucose can thus leave the liver and enter the blood.
Note: glucose-6 phosphatase is involved in both gluconeogenesis and
glycogenolysis.

Fructose-2, 6-bisphosphate:
A. Stimulates glycolysis
B. Is inhibited by PFK1
C. Is inhibited by PFK2
D. Favors gluconeogenesis
E. Stimulates Fructose-1,6-bisphosphatase

Activates glucose
breakdown

Overall Gluconeogenesis reaction


- Leading from pyruvate to free glucose:
2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4H2O

glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+

How do amino acids lead to free


glucose?

Gluconeogenic Substrates
- Lactate (Pyruvate) Cori cycle. Lactate is released into the blood by
muscle, and by cells that lack mitochondria, such as RBCs and taken
to the liver to be used for gluconeogenesis
- Glucogenic amino acids (all, except leucine and lysine).
- Glycerol (from the breakdown of triglycerides in adipose tissue),
-

All TCA intermediates (except acetyl-CoA) are substrates of


gluconeogenesis.

Cori Cycle

After vigorous exercise, lactate produced by anaerobic glycolysis in muscle


returns to the liver and is converted to glucose, which moves back to muscle
and is converted to glycogen a circuit called the Cori cycle.

Cori Cycle: Shuttling of glucose and lactate between muscle and liver
during physical exercise.

Regulation of Gluconeogenesis
Hormones are important for the regulation of gluconeogenesis:

Insulin: is a hormone, released from pancreatic -cells in


response to hyperglycemia (increased blood glucose
level). Insulin lowers the blood glucose level (stimulates
glycogenesis and glycolysis in liver).
Glucagon: is a polypeptide hormone from the -cells of the
pancreas that stimulates the glucose-producing pathways
of the liver (glycogenolysis & gluconeogenesis). It is
released in response to hypoglycemia (decreased blood
glucose level). Glucagon raises the blood glucose level.

Regulation of Gluconeogenesis
Glucagon and insulin secreted by pancreatic and cells,
respectively. The release of glucagon and insulin is inhibited
by somatostatin, which is secreted by the pancreatic
delta cells.

Epinephrine and norepinephrine: are stress hormones that


are released during physical exertion and cold exposure. In the
liver, they favor gluconeogenesis over glycolysis by inducing
cAMP. EPI->G-protein->adenylyl cyclase->cAMP->PKA
->blood glucose
Glucocorticoids: are also stress hormones that affect gene
transcription. Glucocorticoids stimulate gluconeogenesis by
inducing gluconeogenic enzymes.

Regulation of Gluconeogenesis (3)


Pyruvate kinase is the most important regulated enzyme in the
PEP-pyruvate cycle. It is inhibited by ATP and activated by
fructose 1,6-bisphosphate.
Phosphofructokinase and fructose-1,6-bisphosphatase in the liver
are oppositely regulated. ATP and citrate stimulate fructose-1,6bisphosphatase but inhibit phosphofructokinase (favor
gluconeogenesis).

Glucose-phosphorylating enzymes in the liver are


hexokinase/glucokinase the glucose/glucose 6-phosphate reaction.
Just know that when one pathway is activated the other is
inhibited.

Ethanol and Liver Metabolism


-ATP and citrate stimulate fructose 1,6 bisphosphatase but
inhibit phosphofructokinase, means favor
gluconeogenesis.
Ethanol increases both, cytosolic and mitochondrial NADH
+ H+ concentrations. Bad if it uses all the NAD+
- Increased NADH + H+ inhibits the conversion of lactate to
pyruvate, thus inhibiting gluconeogenesis.

Gluconeogenesis
-

Gluconeogenesis takes place almost exclusively in the liver, and the


liver receives large quantities of fatty acids from adipose tissue
during fasting. Insulin inhibits lipolysis, thus in fed state the level of
free fatty acids is low and in fasting state, when insulin is low, FFAs
are high.

Fatty acid oxidation is less controlled by feedback inhibition than is


glucose oxidation. Therefore, the levels of ATP and acetyl-CoA in
the liver are actually elevated during fasting.

- Fatty acid oxidation provides the necessary energy

required by liver for gluconeogenesis. What is the


substrate?
Fatty acid oxidation: The burning of stored fat.

What is the Livers Primary energy


source?

A.Glycolysis
B.Gluconeogenesis
C.Fatty Acid Oxidation
D.A & B

Why Glycogen?
- Fat cannot be as rapidly mobilized in muscle.

- Fat cannot be oxidized to produce energy under


anaerobic conditions.
- Requires energy input to initiate fat oxidation.
- Fat cannot be converted into glucose!
Glycogenesis vs. Glycogenolysis
Liver glycogen only to maintain blood glucose, liver
doesnt use it.

Storage of Glucose and Glycogen


in a normal healthy adult

-Most people have more muscle than liver, the total amount of muscle
glycogen exceeds that in the liver.
There is more glycogen per gram of tissue weight in the liver (it is more
concentrated in the liver)

Glycogen Structure
6

6
5

5
1

4
3

5
1

4
3

Know alpha-1,4- linkages (like starch)


-vs. beta which is cellulose
With alpha-1,6-branching
Add glucose molecules at the reducing end,
take them off at non.

Glycogen Synthesis
Glycogen is readily synthesized from glucose.
(Glucose is a most abundant monosaccharide in dietary carbohydrate)
For glycogen synthesis:
- Primer required to initiate synthesis.
- The protein glycogenin (Mr 37 kDa) serves as the primer,
or that primes the synthesis of new glycogen chains.

- Glycogenin is self glycosylating; attaches glucose from


UDP-glucose to tyrosine residue (Tyr194) of glycogenin.

Glycogen Synthesis

2 high energy bonds

Synthesis of uridine diphosphate (UDP)-glucose. UDP-glucose is the


activated form of glucose, or the precursor for glycogen synthesis.

Which of the following favors glycogen synthesis?


A. Binding of epinephrine to alpha2 receptors
B. The activation of Protein Kinase A and its phosphorylation
cascade
C. Rising levels of AMP in the muscle
D. Activation of glycogen phosphorylase
E. A high insulin/glucagon ratio
What does glycogen phosphorylase do?
122

Glycogen Synthesis
Glycogen synthase is the key regulatory enzyme for glycogen synthesis;
creates the -1,4 glycosidic bonds in glycogen by transferring the
glucose residue from UDP-glucose to non-reducing end of the
glycogen molecule.

Glycogen synthase cannot form the -1,6 glycosidic bonds at the branch
points of glycogen. Branching requires a glycogen-branching
enzyme, called transglycosylase or glycosyl-transferase, that breaks
an -1,4 bond and forms an -1,6 bond .
Glycosidic bonds: are covalent bonds, formed between two
monosaccharide molecules by means of a dehydration reaction.

Which of the following enzymes are not found in


muscle?
A.
B.
C.
D.
E.

Glycogen phosphorylase
Hexokinase
Glucose-6-phosphatase
Phosphoglucomutase
All of the above are found in muscle

In the muscle glycogenolysis is


always coupled to glycolysis!!

Regulation of Glycogen Metabolism


Glycogen metabolism is regulated by hormones and metabolites.
- Glycogen synthesis and glycogen degradation should not be active
at the same time to avoid an ATP-consuming futile cycle, achieved
by the phosphorylation/dephosphorylation of the key enzymes
glycogen synthase and glycogen phosphorylase.
- Glycogen synthase is active in the de-phosphorylated state, whereas
glycogen phosphorylase is active in the phosphorylated state.
Phosphorylation state of the enzymes is regulated by:
- Insulin- Dephosphorylated state both in the liver and in muscle.
- Glucagon- Phosphorylated state (only affects liver)
- Norepinephrine and epinephrine are powerful activators of
glycogen breakdown both in muscle and liver.

Regulation of glycogen synthase and glycogen


phosphorylase

protein kinase phosphorylates both glycogen


phosphorylase and glycogen synthase &
protein phosphatase de-phosphorylates both glycogen
phosphorylase and glycogen synthase .

HORMONE (GLUCAGON / EPINEPHRINE)


Adenylyl
cyclase
ATP

cAMP

C C
R R
Inactive

C C
Active
Kinase A

Protein Kinase A

Ca++ (muscle)
ATP

OH

Inactive
phosphorylase kinase
Phosphoprotein
phosphatase

AMP(muscle)

O-P
Active
Phosphorylase kinase

Glucose

2 ATP
OH OH

OP OP

Phosphorylase b

Phosphorylase a
Phosphoprotein
phosphatase

Glycogen

Glucose-1P

128

Glycogen metabolism enzyme deficiencies -> storage diseases

Von-Gierkes disease (type I) Glycogen buildup in liver


Pompe disease (type II) Glycogen buildup in muscle
(heart)
Cori disease
Anderson disease
McArdle disease
Hers disease
Tarui disease

The binding of epinephrine to receptor on liver cells would result


in intracellular increase in:
A. glucose-6-phosphate dehydrogenase.
B. CAMP
C. Glycogen synthetase activity.
D. glycolysis
E. All of the above.

What is the route of energy


mobilization?

Fructose Metabolism in the Liver


Excess fructose is toxic
The liver metabolized fructose faster than it metabolizes
glucose.
Fructose-1-phosphate tends to accumulate because
fructokinase activity exceeds aldolase B activity
Fructose-1-phosphate directs dietary glucose into glycogen synthesis

Fructose phosphorylation ties up inorganic phosphate (Pi),


which impairs oxidative phosphorylation
131

NADPH:
A. Maintains an oxidative environment of the
cytosol
B. Provides electrons for oxidative
phosphorylation
C. Is a coenzyme for fatty acid synthesis
D. Is mainly found in the mitochondrion

PPP two general products:


NADPH for biosynthesis
Ribose for nucleic acid synthesis

Primary Function of the


PPP
Oxidation of glucose-6-phosphate, makes two important products:
NADPH and ribose-5-phosphate
- occurs in the cytosol of the cell: all the enzymes in the PPP are
located in the cytosol.
- provides NADPH, a coenzyme, for redox reactions or reductive
biosynthetic pathways (such as fatty acid and cholesterol synthesis)
and maintenance of reductive environment of the cytosol -the
defense against oxidative damage or stress.
- provides ribose-5-phosphate for nucleotide (purine and
pyrimidine) and nucleic acid biosynthesis and recycles nucleic
acid-derived pentoses for potential energy use.

Which Tissues?
- all tissues:

- Most active in tissues involved in fatty acid and steroid


synthesis (adrenal gland, liver, adipose tissue and
mammary gland).
- Red blood cells (RBCs): maintain their membrane integrity.
Rapidly dividing cells, such as bone marrow, use ribose
5-phosphate to make RNA, DNA, coenzymes as ATP,
NADH, FADH2 and coenzyme A.
.

2 phases
Oxidative Phase: oxidative branch of the PPP is concerned
with the synthesis of NADPH. Glucose-6-phosphate
dehydrogenase catalyzes the rate-limiting step. The reaction
is irreversible, maintains a high NADPH / NADP+ ratio.

Non-oxidative Phase: non-oxidative branch of the PPP links


ribulose 5-phosphate (product of the oxidative branch) to the
glycolytic and gluconeogenic pathways. This reaction is
reversible. The most important enzymes in this reaction, are
transketolase and transaldolase.
-Pentose can be recycled or used.

Pentose Phosphate Pathway (PPP)


Glucose 6-phosphate dehydrogenase (G6PDH) is inhibited
by a high NADPH / NADP+ ratio.
G6PDH is the first, and most important, enzyme of the
pentose phosphate pathway.

The amounts of glucose 6-phosphate dehydrogenase and


phosphogluconate dehydrogenase are increased in the wellfed state, and this effect is mediated by insulin.
Why is this enzyme sometimes an
issue?

Oxidative reactions of PPP


In some organs, the Pentose Phosphate Pathway ends at
this point, and its overall equation is:
Glucose 6-phosphate + 2 NADP+ + H2O

ribose 5-phosphate + 2 NADPH + 2 H+ + CO2

Non-oxidative reactions of PPP


- Pentose phosphates produced in the oxidative phase, are
recycled into glucose 6-phosphate in non-oxidative phase.
-

In a series of reactions or rearrangements of the carbon skeletons:


- six 5-carbon sugar phosphates are converted to five 6-carbon
sugar phosphates.
- produces glucose 6-phosphate and NADPH.

Two enzymes are unique in PPP in inter-conversions of sugars:


1. Transketolase: catalyzes the transfer of 2-carbon from a ketose donor to an
aldose acceptor, forming the 7-carbon product sedoheptulose 7-phosphate.
The remaining 3-carbon fragment is glyceraldehyde 3-phosphate.
2. Transaldolase: a 3-carbon fragment is removed from sedoheptulose
7-phosphate and condensed with glyceraldehyde 3-phosphate, forming
fructose 6-phosphate, and finally convert to glucose 6-phosphate by
phosphohexose isomerase.

Balance Sheet
Summary of the flow of 15 C atoms through Pentose Phosphate
Pathway reactions by which 5-C sugars are converted to 3-C and 6C sugars.
C5 + C5 C3 + C7
C3 + C7 C6 + C4
C5 + C4 C6 + C3
3 C5 2 C6 + C3

(Transketolase)
(Transaldolase)
(Transketolase)
(Overall)

Glucose 6-phosphate may be regenerated from either the 3-C


glyceraldehyde 3-phosphate or the 6-C fructose 6-phosphate, via
enzymes of gluconeogenesis.

Pentose Phosphate Pathway Scenarios


If nucleic acid synthesis is required (e.g., in actively dividing cells),
the product of the oxidative branch, ribose 5-phosphate (5C), is used
and will not enter the non-oxidative branch.
When the cell needs more NADPH than ribose-5-phosphate, both
the oxidative and non-oxidative branches work in series to yield
NADPH (+CO2) and form fructose-6-phosphate and glyceraldehyde3-phosphate. These products are recycled to glucose-6-phosphate
which goes back to feed the oxidative branch of PPP again, and
again (i.e., all carbons of glucose-6-phosphate may convert to CO2).

If energy is also needed, 5C products are recycled to glucose-6phosphate for glycolysis and repeat PPP reactions (e.g. RBCs).

140

Role of Cytosolic NADPH


- Maintain reductive environment in the cytosol of cell (GSH,
glutathione).
- Provide reducing equivalents for anabolic pathways (fat,
cholesterol synthesis).
- Provide electrons for the generation of Reactive Oxygen
Species (ROS). Making peroxides
- In red blood cells, NADPH produced by the pentose
phosphate pathway, is very important in preventing
oxidative damage
- Cytosolic environment must be maintained in a reduced
form to maintain enzyme functionality.
Reducing environment: An environment without oxygen available for
organisms.

Glutathione
- Glutathione (GSH, tripeptide; Glu-Cys-Gly), functions as a
reducing agent.
- Glutathione Peroxidase (GPx) catalyzes degradation of
hydroperoxides by reduction, as two glutathione molecules
(represented as GSH) are oxidized to a disulfide (GSSG).
Only the reduced form of glutathione (GSH) is an antioxidant.
Therefore, the dimeric, oxidized form (GSSG) has to be reduced back
by the enzyme glutathione-reductase (GR). Can be faulty
(GR depends on NADPH from the pentose phosphate pathway)

2 GSH + ROOH

GPx

GSSG + ROH + H2O

GSSG + NADPH + H+

GR

2 GSH + NADP+

NADPH + H+

GS-SG

NADP+

2 GSH

HSProteinHS

Protein

Active enzyme
Denatured enzyme

Glucose 6-phosphate Dehydrogenase


(G6PDH) Deficiency
- Pentose phosphate pathway is the only source of NADPH in
RBCs.
- G6PDH is the first enzyme of the pentose phosphate pathway.

- G6PDH deficiency has a profound effect on the stability of red


cells, and cannot maintain antioxidant defenses.
- The red cells are unable to handle the additional oxidative stress,
after exposure to drugs (sulfonamides, acetanilid, nalidixic acid,
nitrofurantoin) or food items (fava beans).

Glucose 6-phosphate Dehydrogenase


(G6PDH) Deficiency
- Over 400 genetic variants of G6PDH, mostly coding for less
active/stable enzyme.
- The most common human enzyme deficiency; > 400 million
people affected worldwide, with a shortened life span.
- Confers resistance to malaria. -Sickle cell anemia

Major Organs of Metabolism


Four major organs play a dominant role in fuel metabolism:

1.Liver
2.Adipose
3.Muscle, and
4.Brain.

Which of the following enzymes are favored by


insulin?
A. Glycogen synthetase
B. Glycogen phosphorylase
C. Protein Kinase A
D. Cyclic AMP
E. Glucokinase

Insulin

Insulin: is a hormone, secreted by the cells of pancreas, to


lower blood sugar levels. Insulin is needed to convert sugar,
starches, and other food into glucose (blood sugar) or regulates
storage of glycogen in the liver and accelerates oxidation of sugar
in cells.
Insulin stimulates glycolysis and glycogenesis and
inhibits gluconeogenesis and glycogenolysis.
Insulin: Being a protein, insulin is not orally active because
it is destroyed by digestive enzymes, so mostly used as a
i.p. injection.
Its metabolic effects are anabolic, for example, synthesis of
glycogen, triacylglycerols (TAG), and protein.
The most important hormone for growth and development

Insulin is a Satiety Hormone


- Insulin is the hormone of the well-fed state. Its synthesis and release
are stimulated by glucose, and this effect is further increased by amino
acids. Therefore, the plasma level of insulin is highest after a
carbohydrate-rich meal. During fasting, plasma level of insulin falls.
- Insulin stimulates the utilization of nutrients such as glucose, amino
acids, and triglycerides. It diverts excess nutrients into the synthesis
of glycogen, fat, and protein.

- Insulin regulates the metabolism of fat and protein as well as of


carbohydrate. It induces the conversion of excess carbohydrate to fat
by glycolysis and fatty acid synthesis.

Insulin decreases lipolysis


Puts your energy stores away

Glucagon
Glucagon: is a polypeptide hormone secreted by
the -cells of the pancreas that stimulates the
glucose-producing pathways of the liver. It is
released in response to hypoglycemia (decreased
blood glucose Level, < 40 mg/dl). Hypoglycemia is
a medical emergency.
Glucagon raises the blood glucose level.
Glucagon promotes the release of glucose from the
liver.
Glucagon stimulates glycogenolysis and gluconeogenesis
and inhibits glycolysis. in the liver

Glucagon Maintains the Blood


Glucose Level
- Glucagon secretion from the pancreatic cells is increased two to
three fold by hypoglycemia and reduced to half of the basal release by
hyperglycemia.

- Glucagon up-regulates the blood glucose level when dietary


carbohydrate is in short supply. Its actions on glucose metabolism
pathways, are opposite to those of insulin.
- Unlike insulin, glucagon acts exclusively on the liver; it has
negligible effects on adipose tissue, muscle, and other extra-hepatic
tissues.
For Glucagon think of releasing your energy stores like catecholamines

Which of the following tissues depends upon insulin


for the transport of glucose?
A. brain
B. red blood cells
C. liver
D. Muscle
E. All of the above

What transporter does it use?

What is a normal blood glucose level?

a) 55mmol/liter
b) 90mg/dL
c) 5mg/dL
d) 0.5mmol/liter

Unchecked rise in blood glucose following a


meal would result in severe dehydration, and lead
to hyperosmolar coma.

Fasted State (Liver)


In the fasting state, Glucagon levels are high.
Activation of:

F-2,6 bisphosphatase

Inhibition of:

(loss of fructose-2,6-bisphosphate)

PFK-2

Phosphorylase kinase

Pyruvate kinase

Glycogen phosphorylase

Glycogen synthase

Hormone-sensitive lipase

- The net result is the inhibition of glycolysis and glycogenesis and


- Activation of gluconeogenesis and glycogenolysis.

Fasted State (Liver)


- Four hours after a meal, the liver becomes a net producer of glucose.
Glucose is initially formed by glycogen breakdown, but liver glycogen
lasts for < 1 day.
- During more extended fasting, humans depend entirely on gluconeogenesis to produce ~160 g of glucose per day. At least one half
of this is consumed by the brain.
- Liver supplies glucose and ketone bodies (such as acetoacetate and
-hydroxybutyrate) during fasting.
- Liver first uses glycogen degradation and then gluconeogenesis to
maintain blood glucose levels.
- Under fasting conditions, glucagon is elevated and stimulates
gluconeogenesis.

Prolonged Fast / Starvation


- Major adjustment in tissue glucose utilization occurs:
- Brain and muscle adapt to use ketone bodies and fatty acids thus
decreasing the demand for hepatic gluconeogenesis.
- This spares protein; energy requirement is satisfied by fatty acid
oxidation.

- Red cells remain the major glucose utilizers.

Re-feeding
The levels of glycolytic enzymes in the liver are very low, and
patients severely starved show profound carbohydrate intolerance.
Therefore, re-feeding should be started slowly.

Ethanol and Liver Metabolism


- Alcohol is metabolized in the liver by two oxidation reactions:
1 Ethanol is first converted to acetaldehyde by alcohol dehydrogenase
in the cytosol, and produces NADH (& uses up cytosolic NAD+).
(The capacity to metabolize ethanol, is thus dependent upon the ability
to shuttle NADH into mitochondria (to regenerate NAD+).

2. Acetaldehyde enters mitochondria and is subsequently oxidized to


acetate by aldehyde dehydrogenase and generates NADH. (Note:
This enzyme is inhibited by disulfiram (antabuse), a drug that has
found use in patients desiring to stop alcohol ingestion.
- Increased NADH inhibits gluconeogenesis and fatty acid oxidation.

Reversal of Glucagon action by Insulin


(Re-feeding)
Inhibition of:

Activation of:

- F-2,6 bisphosphatase

- PFK2

- Phosphorylase kinase

- Pyruvate kinase

- Glycogen phosphorylase

- Glycogen synthase

- Hormone-sensitive lipase

Diabetes is caused by Insulin Deficiency or


Insulin Resistance
Type 1 diabetes (insulin-dependent): usually starts in childhood.
It is an autoimmune disease that leads to the destruction of pancreatic
cells. Without endogenous insulin production, the patients depend on
insulin injections for life. Type 1 diabetes afflicts 1 per 400 individuals.

Type 2 diabetes (insulin-resistant): was a disease of middle-aged

and older individuals. It is more common than type 1, is less severe. The
pancreatic cells are intact, and the plasma level of insulin may be
normal, reduced, or elevated. The problem may be either reduced insulin
secretion or insulin-resistance of the target tissues, or a combination of
both.

Diabetes is caused by Insulin


Deficiency or Insulin Resistance
The complete absence of insulin in type 1 diabetes leads to:

- Diabetic ketoacidosis, with severe ketonemia, acidosis, and


blood glucose levels as high as 1000 mg/dL, in which the blood
becomes acidic due to a buildup of ketones.
- Large amounts of glucose and ketone bodies are lost in the
urine, and osmotic diuresis causes dehydration and
electrolyte imbalances. The coma is caused by dehydration,
electrolyte disturbances, and acidosis.
Major ketone bodies: acetoacetate, b-hydroxy butyrate, acetone
Know what ketonuria is

Diabetes is caused by Insulin


Deficiency or Insulin Resistance

- Patients with type 2 diabetes are not afflicted by ketoacidosis b/c they
have insulin to inhibit the breakdown of Fat to acetyl CoA, Acetyl-CoA
doesnt accumulate, so it doesnt get converted to ketones. It is
characterized by excessive glucosuria (glucose in the urine) with
osmotic diuresis (high glucose levels in the urine leads to concentrated
urine, which pulls water into the urine to dilute it) . If the patient
forgets to drink, the resulting dehydration can become sufficiently
severe to affect the central nervous system.
- The over-treatment of diabetes with insulin or oral anti-diabetic drugs
leads to hypoglycemic shock.

Diabetes leads to late complications


Urinalysis is a quick screening test for diabetes

Eighty percent of Type 2 diabetics are obese. Obesity is


the most common cause of insulin resistance.
What are Xenobiotics?
What is polydipsia?

Monooxygenase reaction catalyzed by cytochrome P-450


(CYP) to metabolize foreign chemicals.