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Carbohydrate Metabolism

by:

Dr Hadi Mozafari

8 January 2017 Dr. Mohamed Z Gad 2

A Map of The Major Metabolic Pathways in A Typical Cell

The Diagram was taken from “Biochemistry” by Voet & Voet

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Metabolism involves Catabolic

reactions that break down large, complex molecules to provide energy and smaller molecules.

Anabolic reactions that use ATP energy to build larger molecules.

Metabolism

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Major Pathways of carbohydrate (CHO) Metabolism

CHO metabolism in mammalian cells can be

classified into:

1. Glycolysis: Oxidation of glucose to

pyruvate (aerobic state) or lactate

(anaerobic state)

2. Krebs cycle: After oxidation of pyruvate

to acetyl CoA, acetyl CoA enters the Krebs

cycle for the aim of production of ATP.

3. Hexose monophosphate shunt: Enables

cells to produce ribose-5-phosphate and

NADPH.

4. Glycogenesis: Synthesis of glycogen from

glucose, when glucose levels are high

5. Glycogenolysis: Degradation of glycogen

to glucose when glucose in short supply.

6. Gluconeogenesis: Formation of glucose

from noncarbohydrate sources.

Glucose is the major fuel of most

organisms. The major pathways of CHO

metabolism either begin or end with

glucose.

Glycolysis general

overview

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Glycolysis (Embden-Meyerhof Pathway)

[glycolysis: from the Greek glyk-, sweet, and lysis, splitting]

Glycolysis occurs in cytoplasm of all human cells. Glycolysis do

not need to oxygen and is believed to be among the oldest of all

the biochemical pathways.

Aerobic: Glucose Pyruvate

Anaerobic: Glucose Lactate (or ethanol & acetic acid)

Glycolysis (10 reactions in 2 stages, all in cytoplasm)

1) Endergonic stage:

D-Glucose + 2ATP D-fructose 1,6-biphosphate + 2ADP + 2H+

2) Exergonic stages:

2 D-Glyceraldehyde 3-phosphate + 4ADP + 4Pi + 2H+ 2Lactate + 4ATP

-----------------------------------------------------------------------------

Sum: Glucose + 2ADP + 2Pi ----- 2 Lactate + 2ATP + 2H2O (Anaerobic) Glucose + 2ADP + 2Pi + 2NAD+ ---- 2 pyruvate + 2ATP + 2NADH + 2H+ + 2H2O (Aerobic)

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Common Abbreviations & Alternative Names

Glycolysis Sites:

1. Tissues with no mitochondria: mature RBCs, cornea and lens.

2. Tissues with few mitochondria: Testis, leucocytes, medulla of the kidney, retina, skin

and gastrointestinal tract.

3. Tissues undergo frequent oxygen lack: skeletal muscles especially during exercise.

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1) Endergonic Stage • Glucose (and other hexoses) are

phosphorylated immediately upon

entry into the cell. Phosphorylation

prevents transport of glucose out of

the cell and increases the reactivity

of oxygen in the resulting phosphate

ester.

• Several isoenzymes of hexokinase

with different Km values for glucose

are located in different tissues. Brain

hexokinase has a particularly low

Km for glucose.

• The major enzyme for

phosphorylating glucose in liver is

glucokinase.

• Steps catalyzed by hexokinase &

PFK-1 are irreversible.

Or Glucokinase

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Differences between Glucokinase & Hexokinase

Hexokinase Glucokinase

Present in all tissues Liver only

Low Km for glucose Higher Km for glucose

Strongly inhibited by G6P

(indirectly, save Phosphor)

Not inhibited by G6P (but, inhibited

by F6P)

Non-inducible enzyme, not

affected by diabetes or

insulin

Inducible, synthesis induced by

insulin & repressed in diabetes

Level of enzyme is not

affected by fasting or high

CHO diet

Depends on glucose concentration

Act on glucose, fructose and

galactose

Glucose only

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Glucokinase vs. Hexokinase

Glucokinase: Km = 10 mM,

not inhibited by glucose 6-

phosphate. Present in liver

and in pancreas b cells.

Hexokinase: Km= 0.2 mM,

inhibited by glucose 6-

phosphate. Present in most

cells.

Michaelis-Menten kinetics

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Glucokinase activity is regulated by translocation of the

enzyme between the cytoplasm and the nucleus.

• The reaction catalysed by aldolase is

the reverse of aldol condensation.

• Although the cleavage of F1,6BP is

energetically unfavourable, rapid

removal of the product drives the

reaction forward.

• Of the two products of the aldolase

reaction, only GAP (or G3P) serves as a

substrate for the next reaction in

glycolysis.

• To prevent the loss of the other three-

carbon unit, triose phosphate isomerase

catalyses the interconversion of DHAP

& G3P. Because of this reaction, the

original molecule of glucose has now

been converted to two molecules of

G3P. 15

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2) Exergonic Stage

• G-3-P dehydrogenase is a tetramer,

each subunit contains 1 binding site

for G3P & another for NAD+ (NAD+

is permanently bound to the enzyme).

• G3P 1,3-BPG 3PG and PEP

Pyruvate are examples of “substrate–

level” phosphorylation.

• 3-PG 2-PG is mediated by an

intermediate [2,3-BPG]. Most cells

have low amounts of 2,3-BPG except

in RBCs, which act as allosteric

modifier of Hb-O2 binding.

• PEP Pyruvate is an “irreversible

reaction” due to free energy loss

associated with tautomerization of the

enol to the more stable keto form.

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Catalytic mechanism of

Glyceraldehyde 3-phosphate dehydrogenas

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Mechanism for inactivation of Glyceraldehyde

3-phosphate dehydrogenase by sulfhydryl reagents

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In Erythrocytes, the First Site in Glycolysis for ATP

Formation May Be Bypassed and produce 2,3-BPG

• 2,3-bisphosphoglycerate, which

binds to hemoglobin, decreasing its

affinity for oxygen, and so making

oxygen more readily available to

tissues

GLYCOLYSIS IN ERYTHROCYTE

Erythrocyte lack mitochondria respiratory chain and Kreb’s cycle are absence

Always terminates in lactate

In mammals the reaction catalyzed by phosphoglycerate kinase may be bypassed by a process that catalyzed Biphosphoglycerate mutase

Its does serve to provide 2,3-biphosphoglycerate

bind to hemoglobin decreasing its affinity for oxygen oxygen readily available to tissues

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ADP is converted to ATP by the direct

transfer of a phosphoryl group from a high

energy compound.

Study Question ?????

What is meant by “substrate–

level phosphorylation” ?

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Study Question ?????

Why glycolysis under

anaerobic conditions proceed

to lactate and not just stop at

pyruvate formation ?

The reaction of lactate dehydrogenase is essential in

anaerobic glycolysis , as it is the mean for reoxidizing

NADH formed in the G-3-P dehydrogenase step to re-

enter into the glycolysis cycle. In aerobic glycolysis

reoxidation takes place in mitochondria by the respiratory

chain. Also, glycolysis could be continue.

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Overall pathway of Glycolysis & ATP Formation

Number of ATP generated from

glycolysis

Anaerobic Aerobic Enzyme

-1 ATP -1 ATP Hexokinase

-1 ATP -1 ATP PFK-1

---- +5 ATP G-3-P

dehydrog.

+2 ATP +2 ATP Phospho-

glycerate

kinase

+2 ATP +2 ATP Pyruvate

kinase

+2 ATP +7 ATP Sum

The reason behind this phenomenon is that complete oxidation of glucose under

aerobic conditions yield much more ATP (~38 ATP) than anaerobic glycolysis

(~2ATP). Thus it is anticipated that the rate of glucose consumption will be 19-

20 times faster under anaerobic condition to meet the metabolic demand in a

way equivalent to aerobic conditions. Therefore, due to Pasteur effect glucose

could be saved and acid lactic production inhibited 25

Study Question ?????

Louis Pasteur, the great 19th century French

chemist and microbiologist, was the first scientist

to observe the following phenomenon. “Cells that

can oxidize glucose completely to CO2 and H2O

utilize glucose more rapidly in the absence of O2

than in its presence”. It would appear that O2

inhibits glucose consumption (Pasteur Effect).

Thus, another definition for Pasteur effect is:

inhibition of glucose utilization and lactate

accumulation by the initiation of respiration (O2

consumption)… Can you explain why ?

Louis Pasteur

(1822 - 1895)

• PFK-1 is the major regulatory

enzyme of glycolysis. In the liver

only, PFK-1 is activated by fructose-

2,6-diphosphate (F-2,6-DP).

• PFK-2, the enzyme that synthesize

the activator F-2,6-DP, is itself a

regulatory enzyme. It is inhibited by

citrate & ATP and by

phosphorylation. The reverse reaction

is catalyzed by fructose-2,6-

diphosphatase(F-2,6-DPase).

• Hormones also regulate glycolysis

e.g., glucagon inhibits glycolysis by

repressing the synthesis of F-2,6-DP.

Insulin promotes glycolysis by

stimulating the synthesis of F-2,6-DP. 26

Regulation of Glycolysis

Rate of glycolysis is controlled primarily by

allosteric regulation of the 3 key enzymes

(irreversible steps), hexokinase, PFK-1, and

pyruvate kinase.

Inhibitor Activator Enzyme

G-6-P AMP, ADP, Pi Hexokinase

NADH, H+,

citrate, ATP

F-6-P, AMP,

F-2,6-DP

(liver only)

PFK-1

ATP, acetyl

CoA,

phosphorylation

AMP, F-1,6-

DP

Pyruvate

kinase

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Control points in glycolysis

Types of regulations Mechanism Example

Substrate concentration Saturation kinetics

(Michaelis-Menten

equation)

Glucokinase (activation after a

meal - high Km)

Allosterically A conformational change

after an allosteric activator

binding

Enzymes of glycolysis and

gluconeogenesis (allosteric

efectors: ATP, AMP, citrate)

Covalent modification A conformational change

after phosphorylation by

a protein kinase

Phosphorylation of glycogen

synthase and glycogen

phosphorylase (glucagon)

Protein-protein

interaction

A conformational change

after a modulator protein

binding

Muscle glycogen

phosphorylase (activation by

Ca2+-calmodulin)

Zymogen cleavage Activation by proteolysis of

a precursor molecule

Blood clotting proteins

Enzyme synthesis Induction or represion of

enzyme synthesis

Enzymes of gluconeogenesis

(induction during fasting)

Regulation of enzymes:

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Phosphofructokinase (PFK-1) as a regulator of

glycolysis

fructose-6-phosphate fructose-1,6-bisphosphate PFK-1

PFK allosterically inhibited by:

• High ATP lower affinity for fructose-6-

phosphate by binding to a regulatory site

distinct from catalytic site.

• High H+ reduced activity to prevent

excessive lactic acid formation and drop in

blood pH (acidosis).

• Citrate prevents glycolysis by

accumulation of this citric acid cycle

intermediate to signal ample biosynthetic

precursors and availability of fatty acids or

ketone bodies for oxidation.

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Study Question ?????

What effects do fluoride and

magnesium have on glycolysis ?

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Comments on Glycolysis Glycolysis is the only pathway that produce ATP in absence of O2.

The best known inhibitors of the glycolytic pathway include:

2-Deoxyglucose: causes inhibition of hexokinase.

Sulfhydryl reagents (e.g. Hg-compounds and alkylating agents as

iodoacetate); inhibit glyceraldehydes-3-phosphate dehydrogenase

which has cysteine residue in the active site.

Fluoride: a potent inhibitor of enolase. Thus, fluoride is usually added

to blood samples to inhibit glycolysis before estimation of blood

glucose.

Magnesium: required for kinase reactions by forming Mg-ATP

complex.

Accumulation of lactate is responsible for muscle fatigue and cramps

observed under heavy exercise (anaerobic glycolysis).

Three main regulatory enzymes of glycolysis are: Hexokinase,

Phosphofructokinase & Pyruvate kinase

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Oxidative decarboxylation of

pyruvate by the pyruvate

dehydrogenase complex.

Lipoic acid is joined by an

amide link to a lysine residue

of the transacetylase

component of the enzyme

complex. It forms a long

flexible arm, allowing the

lipoic acid prosthetic group

to rotate sequentially

between the active sites of

each of the enzymes of the

complex

Pyruvate Dehydrogenase (PDH) Complex

PDH complex regulation

Clinical Aspects Inhibition of Pyruvate Metabolism Leads to

Lactic Acidosis

Arsenite and mercuric ions react with the —SH groups of lipoic acid and inhibit pyruvate dehydrogenase, as does a dietary deficiency of thiamin, allowing pyruvate to accumulate.

Many alcoholics are thiamin-deficient (both because of a poor diet and also because alcohol inhibits thiamin absorption), and may develop potentially fatal pyruvic and lactic acidosis. Patients with inherited pyruvate dehydrogenase deficiency also present with lactic acidosis, particularly after a glucose load. PDH deficiency is associated with neurologic disturbances.

Inherited aldolase A deficiency and pyruvate kinase deficiency in erythrocytes cause hemolytic anemia (due to the role of ATP in Na+ /K+

ATPase pump that, maintain the shape of RBC)

The exercise capacity of patients with muscle phosphofructokinase deficiency is low. By providing lipid as an alternative fuel work capacity is improved.

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Krebs Cycle (Citric Acid Cycle)

Cyclic & Amphibolic pathway in mitochondria

The Citric Acid Cycle:

The Catabolism of Acetyl-CoA

The citric acid cycle is the final common pathway

for the aerobic oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino acids are metabolized to acetyl-CoA or intermediates of the cycle.

It also has a central role in gluconeogenesis,

lipogenesis, and interconversion of amino acids.

Oxidative Phosphorylation

- The Citric Acid Cycle Provides

Substrate for the Respiratory Chain

- For Energy & Heat production

- Inhibit by CO, Cyanide &

Thyroid Hormones

- 10 ATP is produced in each round

- Metabolic pathways are Regulated

- Regulatory Enzymes:

A) Slow Quantitative Regulation

- Gene Expression & mRNA

production in long time

• Reactions of the Citric Acid Cycle

Liberate Reducing Equivalents &

CO2

• Vitamins Play Key Roles in the

Citric Acid Cycle:

-Riboflavin

- Niacin

- Thiamin

- Pantothenic acid

Energy production from Glucose

+7ATP

+5ATP

+20ATP

Totally= +32ATP

The Citric Acid Cycle Plays a Pivotal Role in Metabolism

For example:

Transamination and

deamination of amino acids,

and providing the substrates

for amino acid synthesis by

transamination, as well as for

gluconeogenesis and fatty

acid synthesis. Because it

functions in both oxidative

and synthetic processes, it is

amphibolic

The Citric Acid Cycle Take Part in Fatty Acid Synthesis

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Regulation of the Citric Acid Cycle Depends Primarily on a Supply of

Oxidized Cofactors

NAD+ & ADP availability

Citrate synthase, ICD & α-kg dehydrogenase are main regulatory enzymes and stimulates by Ca2+

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Animation & quiz for Krebs Cycle

Krebs Animation

Quiz

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