Glycolysis Part 2BCH 340 lecture 4

There are three steps in glycolysis that have enzymes which regulate the flux of glycolysis

These enzymes catalyzes irreversible reactions of glycolysis

Regulation of Glycolysis

I. The hexokinase (HK)II. The phoshofructokinase (PFK)III. The pyruvate kinase

They are regulatory enzymes which are regulated by the level of ATP in the cell

The most important regulatory enzyme which catalyzes the first irreversible reaction unique to the glycolytic pathway (the committed step)

Allosteric enzyme inhibited by elevated level of ATP, which: is the end product of glycolysis as well as it is substrate for PFK-1

I- Phosphofructokinase-1 (PFK-1):

o Sigmoidal dependence of reaction rate on [fructose-6-P] is seen.

o At high [ATP], PFK has lower affinity for the other substrate, fructose-6-P. ATP binds to inhibition site of PFK, and thereby decreases the activity of enzyme.

I- Phosphofructokinase-1 (PFK-1):

AMP, present at significant levels only when there is extensive ATP hydrolysis, antagonizes effects of high ATP.

AMP, ADP and Fructose 2, 6 biphosphate act as allosteric activators of PFK-1.

I- Phosphofructokinase-1 (PFK-1):

It is allosterically inhibited by its product Glucose 6 phosphate.

In liver, glucokinase is inhibited by Fructose 6P and ATP (acts as a competitive inhibitor of this enzyme)

II- Hexokinase

It is allosterically inhibited by ATP. ATP binding to the inhibitor site of PKdecreases its ability to bind to PEP the substrate.

It is also inhibited by Acetyl Coenzyme A and long chain fatty acid because they are source rich ATP which inhibits PK.

III- Pyruvate Kinase

Insulin is secreted in hyperglycemia and after carbohydrates feeding, it causes:

1. Induction for synthesis of glycolytic key enzyme

2. Activation of protein phosphatase 1 producing dephosphorylation and activation of glycolytickey enzymes

Insulin and Glucagon (secreted by the pancreas) are the main endocrine that modulate blood glucose levels and they act antagonistically

Hormonal regulation of glycolysis

Glucagon is secreted in hypoglycemia or in CHO deficiency and it affects liver cells mainly as follows:

1. It acts as repressor of glycolytic key enzymes (PFK1, Pyruvate kinase, glucokinase)

2. It produces phosphorylation of specific enzymes leading to inactivation of glycolytic key enzymes

Hormonal regulation of glycolysis

Hormonal regulation of glycolysis

Inhibitors of glycolysis

2-deoxyglucose: inhibits hexokinase

Mercury and iodoacetate: inhibit glyceraldehyde-3-P dehydrogenase

Fluoride: inhibits enolase by removal of Mg2+ as Mg fluoride

Arsenate: is uncoupler of oxidation and phosphorylation, it forms 1-arseno-3-phosphoglycerate which interferes with ATP formation at substrate level

Pasteur Effect

It is the inhibition of glycolysis by the presence of

oxygen

Explanation: Aerobic oxidation of glucose produces

increased amount of ATP and citrate. Those inhibit

PFK1.

Mitochondrial pathway for glucose oxidation (TCA cycle)

BCH 340 lecture 5

Under aerobic conditions , pyruvate (the product of glycolysis) passes by special pyruvate transporter into mitochondria which proceeds as follows:

1. Oxidative decarboxylation of pyruvate into acetyl CoA.

2. Acetyl CoA is then oxidized completely to CO2, H2O

through Krebs' cycle

G Pyr

cytosol Mitochodria

glycolyticpathway

secondstage

thirdstage

CO2 + H2O+ATPPyr CH3CO~SCoA

firststage

TAC

Irreversible reaction catalyzed by a multi enzyme

complex associated within the inner mitochondrial

membrane known as Pyruvate dehydrogenase

complex

Oxidative decarboxylation of Pyruvate to Acetyl CoA

COO-

C

CH3

NAD+ NADH + H +

O

pyruvate

CH3CPyruvate

dehydrogenasecomplex

Acetyl CoA

O

~SCoA+ HSCoA + CO2

HSCoA

NAD+

This enzyme complex contains 3 subunits, which catalyze the reaction in 3 steps:

E1 pyruvate dehydrogenase

E2 dihydrolipoyl transacetylase

E3 dihydrolipoyl dehydrogenase

Pyruvate dehydrogenase complex

Es

HSCoA

NAD+

This enzyme needs 5 coenzymes (all are vitamin B complex derivatives)

Pyruvate dehydrogenase complex

Thiamine pyrophosphate, TPP (VB1)

HSCoA (pantothenic acid)

cofactors lipoic Acid

NAD+

FAD (VB2)

Pyruvate dehydrogenase(active form)

allosteric inhibitors:

ATP, acetyl CoA,NADH, FA

allosteric activators:

AMP, CoA,

NAD+,Ca2+

pyruvate dehydrogenase (inactive form)

P

pyruvate dehydrogenase kinase

pyruvate dehydrogenase phosphatase

ATP

ADPH2O

Pi

Ca2+,insulin acetyl CoA,NADH

ADP,

NAD+

Regulation of Pyruvate dehydrogenase complex

Low levels E:E and product accumulation:

(Active dephosphorylated form)

(Inactive phosphorylated form)

1 2

Regulation of E1 by covalent modification through phosphorylation

3

Regulation of Pyruvate Dehydrogenase

Irreversible reaction must be tightly controlled-- three ways

Allosteric Inhibition

Inhibited by products: acetyl-CoA and NADH

Inhibited by high ATP

Allosteric activation by AMP

Ratio ATP/AMP important

Covalent modification (hormonal regulation):

Through Phosphorylation/dephosphorylation of E1

PDH exists in two forms:

Phosphorylated (inactive): Protein kinase enzyme

converts active into inactive enzyme

Dephosphorylated (active): Phosphatase

enzyme converts inactive into active

NB: In vitro inhibition of PDH:• Arsenic• Mercury

Acetyl CoA

cholesterolsynthesis

Cholesterolsteroidhormones

(endocrine glands)

Figure: Metabolic sources and fates

of acetyl CoA

GLUCOSE

PYRUVATE

glycolysis

pyruvatedehydrogenase

lipogenesis

-oxidation

Fatty acids

CO2

citric acidcycle

ketoneoxidation

ketogenesis(liver only)

Ketone bodies

(Cytoplasm)

In mammals, acetyl CoA is essential to the balance between CHO and fat metabolism

Acetyl CoA is an important molecule in metabolism used in many biochemical reactions

Acetyl CoA functions as:

1. input to Krebs Cycle, where the acetate moiety is further degraded to CO2

2. donor of acetate for synthesis of FA, ketone bodies, & cholesterol

Figure: Metabolic sources and fates

of pyruvate and acetyl CoA

GLUCOSEgluconeogenesis

PYRUVATE

glycolysis

Lactate

lactatedehydrogenase

Alanine

alanineamino-transferase

Oxaloacetate

pyruvatecarboxylase

Acetyl CoA

pyruvatedehydrogenase

lipogenesis

-oxidation

Fatty acids

cholesterolsynthesis

Cholesterolsteroidhormones

(endocrine glands)

CO2

citric acidcycle

ketoneoxidation

ketogenesis(liver only)

Ketone bodies

(Cytoplasm)

Kreb's cycle

Also known as Citric Acid Cycle (CAC)

Or

Tricarboxylic Acid Cycle (TCA)

Or

Catabolism of Acetyl CoA (CAC)

Definition: TCA is a series of enzyme-catalyzed

chemical reactions in which acetyl CoA is oxidized

into CO2, H2O and energy.

Location: Occurs in the matrix of the mitochondrion

= aerobically

o The enzymes of TCA are present in the mitochondrial

matrix either free or attached to the inner surface of the

mitochondrial membrane.

Steps:

o The cycle is started by acetyl CoA (2C) and oxaloacetate (4 C)

to form citrate (6C). It ends by oxaloacetate (4C).

o The difference between the starting compound (6C) and the

ending compound (4C) is 2 carbons that are removed in the

form of 2 CO2. These 2 carbons are derived from acetyl CoA.

For this reason acetyl CoA is completely

catabolized in TCA and never gives glucose.

Non-equilibrium reaction catalyzed by citrate synthase

Inhibited by:• ATP

• NADH

• Citrate - competitive inhibitor of oxaloacetate

The cycle begins with the condensation of acetyl-CoA and oxaloacetate to form citrate

Equilibrium reactions

Results in interchange of H and OH

Aconitase then catalyzes the interconversion of citrate and isocitrate via dehydration and hydration

Isocitrate is then converted to α-ketoglutarate via oxidative decarboxylation, producing CO2

Isocitrate dehydrogenated and decarboxylated to give -

ketoglutarate

Non-equilibrium reaction catalyzed by isocitrate dehydrogenase

NAD+

Isocitrate is then converted to α-ketoglutarate via oxidative decarboxylation, producing CO2

Results in formation of:

o NADH + H+

o CO2

Stimulated by isocitrate, NAD+, Mg2+, ADP, Ca2+

Inhibited by NADH and ATP

NAD+

TPP lipoate

FAD

The α-ketoglutarate is then converted to succinyl-CoA via another oxidative decarboxylation, producing the second CO2

Series of reactions result in decarboxylation, dehydrogenation and

incorporation of CoASH

Non-equilibrium reactions catalyzed by -ketoglutarate dehydrogenase

complex

Stimulated by Ca2+

Inhibited by NADH, ATP, Succinyl CoA

Equilibrium reaction catalyzed by succinate thiokinase

Results in formation of GTP and CoA-SH

Nucleoside diphosphate kinase interconverts GTP and ATP

by a readily reversible phosphoryl transfer reaction: GTP +

ADP ↔ GDP + ATP

Succinyl CoA is then converted to succinate, accompanied by the formation of a GTP (or ATP)

Succinate dehydrogenated to form fumarate

Equilibrium reaction catalyzed by succinate dehydrogenase

–Only Krebs enzyme contained within inner mitochondrial membrane

Results in formation of FADH2

Succinate is then converted to fumarate by dehydrogenation

Equilibrium reaction catalyzed by fumarase

Fumarate is then converted to malate via hydration

The cycle ends by the regeneration of oxaloacetate from L-malate

Malate dehydrogenated to form oxaloacetate

Equilibrium reaction catalyzed by malate dehydrogenase

Results in formation of NADH + H+

Acetyl CoAPyruvate

Oxaloacetate

fatty acids, ketone bodies

PDH

Glucose

glycolysis

CoA

Citrate

cis Aconitate

Isocitrate

-Ketoglutarate

NAD+

NADH, CO2

Fumarate

FAD

FADH2

Malate

NAD+

NADH

Succinate GDP

GTP

ADP

ATP

CoA, NAD+

Succinyl CoANADH, CO2

Figure: Reactions of the citric acid cycle

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Products of Krebs Cycle

2 CO2

3 NADH

1 ATP

1 FADH2

Each NADH energizes 3 ATP

Each FADH2 energizes 2 ATP

Double this list for each glucose

ATP Yield

By transamination, oxaloacetate is converted to aspartate

The amphibolic nature of Citric acid cycle

This pathway is utilized for the both catabolic reactions to generate energy as well as for anabolic reactions to generate metabolic

intermediates for biosynthesis

By transaminationα-Ketoglutarate is converted to glutamate

What are the key regulated enzymes in citrate cycle?

Pyruvate dehydrogenase – not a citrate cycle enzyme but it is critical to flux of acetyl-CoA through the cycle; this multisubunitenzyme complex is inhibited by acetyl-CoA, ATP and NADH.

What are the key regulated enzymes in citrate cycle?

Citrate synthase – catalyzes the first reaction in the pathway and can be inhibited by citrate, succinyl-CoA, NADH and ATP; inhibition by ATP is reversed by ADP.

Isocitrate dehydrogenase - catalyzes the oxidative decarboxylation of isocitrate by transferring two electrons to NAD+ to form NADH, and in the process, releasing CO2, it is activated by ADP and Ca2+ and inhibited by NADH and ATP

What are the key regulated enzymes in citrate cycle? (Cond…)

α-ketoglutarate dehydrogenase - functionally similar to pyruvatedehydrogenase in that it is a multisubunit complex, requires the same five coenzymes and catalyzes an oxidative decarboxylationreaction that produces CO2, NADH and succinyl-CoA; it is activated by Ca2+ and AMP and it is inhibited by NADH, succinyl-CoA and ATP

Inhibitors of TCA

Fluoroacetyl CoA: it combines with oxaloacetate giving rise to fluorocitrate which inhibits aconitase enzyme

Malonic acid: inhibits succinate dehydrogenase (competitive inhibition)

Arsenate and Mercury : inhibit Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complex by reacting with sulphydral group of lipoic acid leading to accumulation of pyruvic lactic acid and α-ketoglutarate with acidosis