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Chapter 18

Glycolysis

Dr Khairul Ansari

Chapter 18

Living organisms, like

machines, conform to the law

of conservation of energy, and

must pay for all their activities

in the currency of catabolism.

Ernest Baldwin

Dynamic Aspects of

Biochemistry

Louie Pasteur s scientific

investigations into

fermentation of grape sugar

were pioneering studies of

glycolysis.

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What is the chemical basis and logic for glycolysis, the centralpathway of metabolism; that is, how does glycolysis work?

What are the essential features of glycolysis?

Why are coupled reactions important in glycolysis?

What are the chemical principles and features of the first phaseof glycolysis?

What are the chemical principles and features of the secondphase of glycolysis?

What are the metabolic fates of NADH and pyruvate producedin glycolysis?

How do cells regulate glycolysis?

Are substrates other than glucose used in glycolysis? How do cells respond to hypoxic stress?

Essential Question

Living organism appears in O2 free environment

Gycolysis does not need O2

Gycolysis plays significant roles in anaerobic metabolism in the

first 2 billion years of evolution

Otto Warburg, Gustav Embden and Otto Fritz Meyerhof- worked

out of the glycolysis pathway

Gycolysis is also known as Embden-Meyerhof (or Warburg)

Pathway

Brain, Kidney medulla and rapidly contracting skeleting muscle

and some cells such as erythrocytes and sperm use glucose as

only energy source.

Gycolysis

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Glycolysis

Essentially all cells carry out glycolysis

Ten reactions essentially the same in all cells but with differentrates

Two phases:

First phase converts glucose to two glyceraldehyde-3-P

Second phase produces two pyruvates

Products are pyruvate, ATP and NADH

Three possible fates for pyruvate

Under aerobic conditions pyruvate can be converted to acetyl-CoA

that enter TCA cycle

It can also be converted into glucose via gluconeogenesis.

Under anaerobic conditions, it may be converted into lactic acid. In

yeast, pyruvate is converted into ethanol instead.

The Fates of Pyruvate From Glycolysis

Figure 18.2 Pyruvate produced in glycolysis can be used by cells in

several ways. In animals, pyruvate is normally converted to acetyl-

coenzyme A, which is then oxidized in the TCA cycle to produce CO2.

When oxygen is limited, pyruvate can be converted to lactate.

Alcoholic fermentation in yeast converts pyruvate to ethanol and CO2.

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The Glycolysis Pathway

Phase I:- Five reaction break down glucose to two molecules of

Glyceraldehyde -3-Phosphate (G-3-P) consume two ATP

Phase II:- G-3-P is converted to two molecules of pyruvate-produce 4

ATP

The Glycolysis Pathway

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Why Are Coupled Reactions Important in Glycolysis?

Coupled reactions convert some, but not all of the metabolic energy

of glucose into ATPUnder cellular conditions, approximately 5% of the energy of glucose

is released in glycolysis

Coupled reactions involving ATP hydrolysis are also used to drive the

glycolytic pathway

Net gain of Glycolysis is 2 ATP (61 kj/mol) and 2 pyruvate

Glucose 2 molecules of lactate

C6H12O6 2 H3C-CHOH-COO- + 2 H+

G = -183.6 KJ/molThe excess energy is utilized in rearrangement of the molecules

Glucose + 2 ADP + 2 P i 2 lactate + 2 ATP + 2 H+ 2 H2O

G = -183.6 + 61 = - 122.6 KJ/molUnder standard condition (61/183.6)x 100 = 33% energy is stored in

ATP

What Are the Chemical Principles and Features of

the First Phase of Glycolysis?

The first reaction - phosphorylation of glucose

Hexokinase or glucokinase

This is a priming reaction - ATP is consumed here in order to getmore later

ATP makes the phosphorylation of glucose spontaneous

Be sure you can interconvert Keq and standard-state free energychange

Be sure you can use Eq. 3.13 to generate the values shown in thefar right column of Table 18.1.

-D-Glucose + ATP4- -D-Glucose -6-phosphate2- +ADP3- + H+

G = -16.7 KJ/molPhosphorylation of glucose cost 13.8 KJ/mol

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Hexokinase Primes the Pump for Glycolysis

Figure 18.3 Just as a water pump

must be primed with water to

get more water out, the glycolytic

pathway is primed with ATP in

steps 1 and 3 in order to achieve

net production of ATP in the

second phase of the pathway.

Advantages of Phosphorylation

1. Plasma membrane is impermeable to -D-Glucose -6-phosphate2-

(negatively charged)2. Conversion of glucose to -D-Glucose -6-phosphate2- , keeps the

intracellular concentration low, favoring diffusing into the cell

3. Favorable thermodynamics of the first reaction makes it an

important site for regulation

Glucose is kept in the cell by phosphorylation to

glucose-6-phosphate

Figure 18.4 Glucose-6-P cannot cross the plasma membrane.

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Reactions and Thermodynamics of Glycolysis

Rxn 1: Hexokinase1st step in glycolysis; G large, negative

Hexokinase (and glucokinase) act to phosphorylate glucose andkeep it in the cell

Km for glucose is 0.1 mM; cell has 4 mM glucose

So hexokinase is normally active!

Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in

glucose

Hexokinase is regulated - allosterically inhibited by (product)glucose-6-P - but is not the most important site of regulation ofglycolysis - Why?

Hexokinase reaction is one of the three points in glycolysispathway that are regulated

Mg2+ is required (the true substrate is MgATP2-)

There are two isozymes of Hexokinase (type I and type II)

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Km for glucose is 0.03 mM for type I hexokinase and 0.3 mM fortype II hexokinase

Type I is primary hexokinase in brain

Type IV (glucokinase) is predominant in liver- doest not get

inhibited by the product (Km is approximately 10 mM)

When glucose level is high the liver hexokinase (glucokinase)

phosphorylates glucose and eventually converts to glycogen

Glucokinase is inducible (by insulin) and acts when level of

glucose is very high.

In diabetes mellitus (patient producing insufficient insulin) level

of glucokinase is less.

Rxn 1: Hexokinase

Steady-State Concentrations of Glycolytic Intermediates

These steady-state concentrations are

used to obtain the cellular values ofG

found in Table 18.1 and Figure 18.22.

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Glucose-6-P is common to several metabolic pathways

Figure 18.5 Glucose-6-phosphate is the branch point for several

metabolic pathways.

Reaction 1: Hexokinase

Figure 18.6 The (a) open and (b) closed

states of yeast hexokinase. Binding of

glucose (green) induces a conformation

change that closes the active site, as

predicted by Daniel Koshland. The induced

fit model for enzymes is discussed on page

409 of the text.

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Hexokinase is the paradigm of induced fit

Figure 18.7 (a) Mammalian

hexokinase I contains an N-

terminal domain (top) and a C-

terminal domain (bottom) joined

by a long -helix. Each of these

domains is similar in sequence

and structure to yeast hexokinase.

(b) Human glucokinase undergoes

induced fit upon binding glucose

(green).

Reaction 2: Phosphoglucoisomerase

Glucose-6-P to Fructose-6-P Why does this reaction occur?

next step (phosphorylation at C-1) would be tough for

hemiacetal -OH, but easy for primary -OH

isomerization activates C-3 for cleavage in aldolase reaction

Ene-diol intermediate in this reaction

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A mechanism for phosphoglucoisomerase

Figure 18.8 The phosphoglucoisomerase mechanisminvolves opening of the pyranose ring (step 1), proton

abstraction leading to enediol formation (step 2), and

proton addition to the double bond, followed by ring

closure (step 3)

Reaction 3: Phosphofructokinase

PFK is the committed step in glycolysis!

The second priming reaction of glycolysis

Committed step and large, negative G - means

PFK is highly regulated

ATP inhibits, AMP reverses inhibition

Citrate is also an allosteric inhibitor

Fructose-2,6-bisphosphate is allosteric activator

PFK increases activity when energy status is low

PFK decreases activity when energy status is high

The reaction is endegonic by itself (G = 16.6 KJ/mol), butexergonic when coupled with ATP hydrolysis (G = -14.2 KJ/mol

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Phosphofructokinase a Second Phosphorylation

Driven by ATP

Phosphofructokinase is the second priming reaction of glycolysis.

ATP is consumed in this priming reaction, so that more ATP can be

produced further along the pathway.

PhosphofructokinasePhosphofructokinase is the valve controlling rate of glycolysis

ATP is both substrate and allosteric regulator of PFK

High ATP turn off glycolysis in cytosol.

ATP level does not fluctuate to greater extend in most tissue

ADP + ADP ATP + AMP

Small drop of ATP level in cell results a large increase in AMP

AMP reverse the PFK inhibition by ATP

PFK activity is high when energy status of cell is low, and PFK

activity is low when energy status is high.

Fructose-2,6-bisphosphate also regulatesPFK

Citrate an intermediate of TCA cycle is also an allosteric inhibitor

of PFK (PFK activity coupled with TCA cycle)

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Phosphofructokinase behaves cooperatively at high [ATP]

Figure 18.9 At high ATP, phosphofructokinase (PFK) behaves

cooperatively and the activity plot is sigmoid.

F-2,6-BP regulates Phosphofructokinase

Phosphofructokinase is regulated by fructose-2,6-bisphosphate, a

potent allosteric activator that increases the affinity of

phosphofructokinase for the substrate fructose-6-P.

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F-2,6-BP reverses the inhibition of PFK by ATP

Figure 18.11 F-2,6-BP

stimulates PFK by

decreasing the inhibitory

effects of ATP.

Rxn 4: Aldolase (Fructose bisphosphate aldolase

A C6

intermediate is cleaved to 2 C3

s (Dihydroxyacetone phosphate

and and Glyceraldehyde-3-phosphate)

The reaction is enderonic (cellularG, however, is close to zero.)

The reaction rate (equilibrium ) is influenced by concentration and

may become exergonic

Class I and II aldolases present in animal and bacteria respectively

Class II aldolase contain Zn2- in the active site

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Reaction 5: Triose Phosphate Isomerase

Triose phosphate isomerase completes the first phase of glycolysis.

Each glucose has been converted to two molecules of glyceraldehyde-

3-phosphate.

Reaction 5: Triose Phosphate Isomerase

DHAP is converted to G-3-P

This reaction makes it possible for both products of the aldolase

reaction to continue in glycolysis

The reaction involves an ene-diol mechanism

Glu165 in the active site acts as a general base

Triose phosphate isomerase is a near-perfect enzyme

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What Are the Chemical Principles and Features of

the Second Phase of Glycolysis?

Metabolic energy of glucose produces 4 ATP

Net ATP yield for glycolysis is two ATP

The second phase of glycolysis involves two very high-energy

phosphate intermediates

1,3-bisphosphoglycerate (1,3-BPG)

Phosphoenolpyruvate (PEP)

Reaction 6: Glyceraldehyde-3-P DehydrogenaseG-3-P is oxidized to 1,3-BPG

Oxidation of aldehyde to carboxylic acid is highly exergonic

The overall reaction involves formation of carboxylic phosphoricanhydride and the reduction of NAD+ to NADH

The overall reaction is slightly endergonic

Energy yield from converting an aldehyde to a carboxylic acid isused to make 1,3-BPG and NADH

The mechanism involves covalent catalysis and a nicotinamidecoenzyme, and it is good example of nicotinamide chemistry

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Rxn 6: Glyceraldehyde-3-P Dehydrogenase

Figure 18.14 A

mechanism for the

glyceraldehyde-3-

phosphate

dehydrogenase reaction.

Reaction of an enzyme

sulfhydryl with G3P forms

a thiohemiacetal, which

loses a hydride to NAD+ to

become a thioester.

Phosphorolysis releases1,3-bisphosphoglycerate.

G3P-DH

Arsenate is a substrate for the G3P-DH reaction, forming1-arseno-3-phosphoglycerate. This product breaks down to

3-phosphoglycerate, essentially bypassing the phosphoglycerate

kinase reaction. The result is that glycolysis in the presence of

arsenate produces no net ATP.

G3P-OH can be inactivated by reaction with iodoacetate, whichreacts with and block the essential cysteine sulfhydryl

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Reaction 7: Phosphoglycerate Kinase

ATP synthesis from a high-energy phosphate Phosphoglycerate kinase transfers a phosphoryl group from 1,3-

bisphosphoglycerate to ADP to form an ATP.

This is referred to as substrate-level phosphorylation

This reaction pays off the ATP debt created by the priming

reactions in the first phase

Mg2+ is required (the true substrate is MgADP-)

2,3-BPG (for hemoglobin) is made by circumventing the PGK

reaction (Figure 18.15)


Phosphoglycerate kinase transfers a phosphoryl group from 1,3-

bisphosphoglycerate to ADP to form ATP. This type of ATP-

synthesizing reaction is referred to as a substrate-level

phosphorylation

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The open (a) and closed (b) forms of

phosphoglycerate kinase. ATP (cyan), 3-

phosphoglycerate (purple), and Mg2+

(gold).

2,3-BPG is made by reactions that detour around the

phosphoglycerate kinase reaction

2,3-bisphosphoglycerate is an important regulator of

hemoglobin (see pages 505-506 of the text)

2,3-BPG is formed from 1,3-BPG by bisphosphoglycerate

mutase

3-phosphoglycerate is then formed by 2,3-

bisphosphoglycerate phosphatase

Most cells contain only a trace of 2,3-BPG, but erythrocytes

typically contain 4-5 mM 2,3-BPG

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phosphoglycerate kinase reaction

Figure 18.15 Formation and decomposition of 2,3-

bisphosphglycerate.


phosphoglycerate kinase rxn

Figure 18.16 The mutase that forms 2,3-BPG from 1,3-BPG requires

3-phosphoglycerate. The reaction is actually an intermolecular

phosphoryl transfer from C-1 of 1,3-BPG to C-2 of 3-

phosphoglycerate.

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Reaction 8: Phosphoglycerate Mutase

Phosphoglycerate mutase catalyzes a phosphoryl group transfer

from C-3 to C-2

Rationale for this reaction in glycolysis: It repositions the

phosphate to make PEP in the following reaction (enolase)

Note the phospho-histidine intermediates

Zelda Rose (wife of Nobel laureate Irwin Rose) showed that a

bit of 2,3-BPG is required to phosphorylate His

Nomenclature note: a mutasecatalyzes migration of a

functional group within a substrate

Reaction 8: Phosphoglycerate Mutase

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Reaction 9: Enolase

Conversion of 2-Phosphoglycerate to PEP

The enolase makes a high-energy phosphate in

preparation for ATP synthesis in step 10

The overall G for this reaction is 1.8 kJ/mol

How can such a reaction create a PEP?

"Energy content" of 2-PG and PEP are similar

Enolase just rearranges 2-PG to a form that releases

more energy upon hydrolysis

Reaction 9: Enolase

The enolase reaction creates a high-energy phosphate in

preparation for ATP synthesis in step 10 of glycolysis.

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Reaction 10: Pyruvate Kinase

Conversion of PEP to pyruvate makes ATP

These two ATP (from one glucose) can be viewed as the"payoff" of glycolysis

Large, negative G indicating that this reaction issubject to regulation

PK is allosterically activated by AMP, F-1,6-bisP

PK is allosterically inhibited by ATP and acetyl-CoA

Understand the keto-enol equilibrium of pyruvate; it is

the key to understanding the pyruvate kinase reaction


The pyruvate kinase reaction converts PEP to pyruvate, driving

synthesis of ATP. Note that two ATP are produced here, since two

PEP are formed from one glucose. These two ATP are the payoffof glycolysis.

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Figure 18.19 The conversion of phosphoenolpyruvate (PEP) topyruvate may be viewed as involving two steps: phosphoryl transfer,

followed by an enol-keto tautomerization. The tautomerization is

spontaneous and accounts for much of the free energy change for

PEP hydrolysis.

18.5 What Are the Metabolic Fates of NADH and Pyruvate

Produced in Glycolysis?

NADH can be recycled via aerobic or anaerobic pathways

NADH represents energy - two possible fates:

If O2 is available (aerobic conditions), NADH isoxidized in the electron transport pathway, makingATP in oxidative phosphorylation

In anaerobic conditions, NADH is oxidized bylactate dehydrogenase (LDH), providing additionalNAD+ for more glycolysis

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Figure 18.21 (a) Pyruvate reduction to ethanol in yeast provides a means for

regenerating NAD+ consumed in the glyceraldehyde-3-P dehydrogenase reaction.

(Right) Fermentation at a bourbon distillery. A mash of corn and other grains is

fermented by yeast, producing ethanol and CO2, which can be seen bubbling to the

surface.



Figure 18.21 (b) In oxygen-depleted muscle, NAD+ is regenerated in the

lactate dehydrogenase reaction. Hibernating turtles, trapped beneath

ice and lying in mud, become anoxic and convert glucose mainly to

lactate. Their shells release minerals to buffer the lactate throughout

the period of hibernation.

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Pyruvate also represents energy - two possiblefates:

Aerobic or anaerobic paths

In aerobic conditions, pyruvate proceeds throughthe tricarboxylic acid (TCA) cycle (see Chapter 19)

Anaerobic metabolism of pyruvate leads to lactate(in microorganisms and animals) or ethanol (inyeast)

These are examples offermentation theproduction of ATP energy by reaction pathways inwhich organic molecules function as donors andacceptors of electrons

The Warburg Effect and Cancer

Otto Warburg observed in 1924 that rapidlyproliferating cancer cells metabolize glucose mainly tolactate, even when O2 is plentiful

Lewis Cantley has suggested that this behavior arisesbecause cells need more than ATP they mustsynthesize large amounts of nucleotides, amino acids,and lipids

This requires lots of NADPH for biosynthesis as well asintermediates for building blocks

Cancer cells divert large amounts of glucose to thepentose phosphate pathway to produce NADPH

See page 597 for more details

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Otto Warburg

Otto Warburg, Nobel Prize in

Physiology or Medicine, 1931

for his discovery of the nature

and mode of action of the

respiratory enzyme

(cytochrome oxidase see Ch.

20)

The Warburg Effect and Cancer

Signaling proteins and

pathways regulate the

glycolytic pathway. Cancer

cells route up to 90% of

acquired glucose and

glutamine into lactate and

alanine, producing large

amounts of NADPH

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18.6 How Do Cells Regulate Glycolysis?

The elegant evidence of regulation See Figure 18.22

Standard state G values are scattered, with both plusand minus values and no apparent pattern

The plot ofG values in cells is revealing:

Most values near zero

3 of 10 reactions have large, negative G

These 3 reactions with large negative G are sites ofregulation (HK, PFK, PK)

Regulation of these three reactions can turn glycolysisoff and on


Figure 18.22 The free

energies of the reactions of

glycolysis under standard-

state conditions.

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Figure 18.22 The free

energies of the reactions of

glycolysis under actual

intracellular concentrations

of metabolites in

erythrocytes.

Tumor Diagnosis Using Positron Emission Tomography (PET)

Tumors show very high rates of glycolysis, as shown by OttoWarburg early in the 20th century

This observation is the basis of tumor detection by positronemission tomography (PET)

Metabolites labeled with 18F can be taken up by humancells (in the brain, for example)

Decay of18F results in positron emission

Positron-electron collisions produce gamma rays

Detection with gamma ray cameras provides 3D models of

tumor extent and location 2-[18F]fluoro-2-deoxy-glucose, used for this purpose, is a

substrate for hexokinase

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Tumor Diagnosis Using Positron Emission Tomography (PET)

2-[18F]fluoro-2-deoxy-

glucose is a substrate for

hexokinase

Decay of18F results in positron

emission.

Positron-electron collisions

produce gamma rays

Detection with gamma ray cameras provides 3D models of

tumor extent and location

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18.7 Are Substrates Other Than Glucose Used in Glycolysis?

Sugars other than glucose can be glycolytic substrates

Fructose, mannose and galactose can all be used

Fructose and mannose are routed into glycolysis by

fairly conventional means. See Figure 18.23

Galactose is more interesting - the Leloir pathway

"converts" galactose to glucose - sort of....

See Figure 18.24


Figure 18.23

Mannose, galactose,

fructose, and other

simple metabolites

can enter the

glycolytic pathway.

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Figure 18.24 Galactose

metabolism via the Leloir

pathway.

Galactose Enters Glycolysis Via the Leloir Pathway

Figure 18.25 The galactose-1-phosphate uridylyltransferase

reaction involves a ping-pong kinetic mechanism.

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UDP-glucose pyrophosphorylase uses Gal-1-P, reducing galactose

toxicity in adultsFigure 18.26

Glycerol Can Also Enter Glycolysis

Glycerol is produced in the decomposition of triacylglycerols. It can

be converted to glycerol-3-P by glycerol kinase. Glycerol-3-P is then

oxidized to dihydroxyacetone phosphate by the action of glycerol

phosphate dehydrogenase.

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Glycerol Can Also Enter Glycolysis

Glycerol is produced in the decomposition of triacylglycerols. It can

be converted to glycerol-3-P by glycerol kinase. Glycerol-3-P is then

oxidized to dehydroxyacetone phosphate by the action of glycerol

phosphate dehydrogenase.

Lactose From Mothers Milk to Yogurt and

Lactose Intolerance

In placental mammals, lactose is synthesize only in themammary gland, and then only during late pregnancyand lactation

The synthesis is done by lactose synthase, a dimericcomplex of galactosyl transferase and -lactalbumin

Lactose breakdown in the intestines by lactaseprovides newborns with essential galactose

Some humans are lactose intolerant, due to a

particularly low level of lactase

Lactic acid fermentation by certain bacteria is the basisfor the production ofyogurt

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Lactose From Mothers Milk to Yogurt and

Lactose Intolerance

Breakdown of lactose to

galactose and glucose by

lactase.

Many Humans are Lactose Intolerant Due to a Low

Level of Lactase

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18.8 How Do Cells Respond to Hypoxic Stress?

Glycolysis is an anaerobic pathway

The tricarboxylic acid cycle is aerobic

When oxygen is abundant, cells prefer to combine thesepathways in aerobic metabolism

When oxygen is limiting, cells adapt to carry out moreglycolysis

Hypoxia causes changes in gene expression that increaseslevels of glycolytic enzymes

A trigger for this is a DNA-binding protein called hypoxiainducible factor (HIF)

HIF is regulated at high oxygen levels by hydroxylase factor-inhibiting HIF (FIH-1)


HIF consists of two subunits: a ubiquitous HIF-1subunit and a hypoxia-responsive HIF-1 subunit

In response to hypoxia, inactivation of the prolylhydroxylases allows HIF-1 stabilization

Dimerization with HIF-1

Binding of the dimer to the hypoxia-responsive element(HRE) of HIF target genes

Activation of transcription of these genes

VHL is the von Hippel Lindau subunit of the ubiquitinE3 ligase that targets proteins for proteasomedegradation

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Figure 18.28

Chapter 19

The Tricarboxylic Acid Cycle

Dr Khairul Ansari

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Chapter 19

Thus times do shift, each

thing his turn does hold; New

things succeed, as former

things grow old.

Robert Herrick

Hesperides (1648)

A time-lapse photograph of a

ferris wheel at night. Aerobiccells use a metabolic wheel

the TCA cycle to generate

energy by acetyl-CoA

oxidation.

Essential Questions

How is pyruvate oxidized under aerobic conditions,

and what is the chemical logic that dictates how

this process occurs?

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Outline

What is the chemical logic of the TCA cycle? How is pyruvate oxidatively decarboxylated to acetyl-CoA?

How are two CO2 molecules produced from acetyl-CoA?

How is oxaloacetate regenerated to complete the TCA cycle?

What are the energetic consequences of the TCA cycle?

Can the TCA cycle provide intermediates for biosynthesis?

What are the anaplerotic, or filling up, reactions?

How is the TCA cycle regulated?

Can any organisms use acetate as their sole carbon source?

Oxidation of glucose to CO2 is a

24-electron oxidation

The electrons from glucose

oxidation feed into the electron

transport pathway, driving

synthesis of ATP.

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Hans Krebs Showed That the Oxidation of Acetate is

Accomplished by a Cycle

The TCA cycle is known as the Krebs cycle and also as the citric acidcycle

Pyruvate from glycolysis is oxidatively decarboxylated to acetate

Then acetate is degraded to CO2 in the TCA cycle

Some ATP is produced

More NADH is made

NADH goes on to make more ATP in electron transport and oxidativephosphorylation

4 electrons are removed as NADH in glycolysis

2 NADH are produced during decarboxylation of two molecules of

pyruvate

For each acetyl-CoA oxidized in TCA cycle, 8 more electrons are

removed as NADH (3) and FADH2(1)

H3CCOO- + 2H20 + H

+ 2CO2 + 8H

In electron transport pathway these electrons combines with O2

to produce water

8H + 2O2 H2O

Net reaction of TCA cycle and electron transport pathway isH3CCOO

- + 2O2 + H+ 2CO2 + 2H2O

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Acetyl-CoA- entry point of new carbon into the TCA cycle

Generally (in biological system) C-C occur cleavage between a

and carbon

Or between two carbons

As acetate does not have a carbon neither of these cleavage are

possible.

Condensation of acetate with oxaloacetate facilitate cleavage

Figure 19.1 The Tricarboxylic Acid Cycle

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-Cleavage of an -Hydroxyketone

This type of cleavage occurs in the transaldolase reaction (described in

Chapter 22).

But it would require hydroxylation of acetate, which is not a favorable

or facile reaction for acetate

Living things have evolved the clever chemistry of condensing acetate

with oxaloacetate and then carrying out a -cleavage TCA combines this cleavage with oxidation to form CO2, regenerating

oxaloacetate and capturing all the energy as NADH and ATP!

19.2 How Is Pyruvate Oxidatively Decarboxylated to Acetyl-

CoA?

Pyruvate must enter the mitochondria to enter the TCA cycle

Oxidative decarboxylation of pyruvate is catalyzed by thepyruvate dehydrogenase complex

Pyruvate dehydrogenase is a noncovalent assembly of threeenzymes

Five coenzymes are required

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Figure 19.4 The Reaction Mechanism of the Pyruvate

Dehydrogenase Complex

Decarboxylation of pyruvate yields hydroxyethyl-TPP. Transfer to lipoic

acid is followed by formation of acetyl-CoA.

19.3 How Are Two CO2 Molecules Produced from Acetyl-

CoA?

The citrate synthase synthase reaction initiates the TCA cycle

Citrate synthase is classic CoA chemistry

C of the acetyl group in acetyl-CoA is acidic and can bedeprotonated to form a carbanion

This carbanion is a strong nucleophile that can attack the -carbonylof oxaloacetate, yielding citryl-CoA

Thioester hydrolysis then produces citrate

NADH & succinyl-CoA are allosteric inhibitors

Note (Table 19.1) that citrate synthase has a large negative G and isa site of regulation

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The citrate synthase reaction initiates the TCA cycle

Figure 19.6 Citrate is formed in the citrate synthase reaction from

oxaloacetate and acetyl-CoA. The mechanism involves nucleophilic

attack by the carbanion of acetyl-CoA on the carbonyl carbon of

oxaloacetate, followed by thioester hydrolysis.

NADH is an allosteric inhibitor of citrate synthase

The citrate synthase synthase reaction initiates the TCA

cycle

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The citrate synthase synthase reaction initiates the TCA cycle

Figure 19.7 Citrate synthase in mammals

is a dimer of 49-kD subunits. In the

monomer shown here, citrate (blue) and

CoA (red) bind to the active site, which lies

in a cleft between two domains and is

surrounded mainly by -helical segments.

Citrate Is Isomerized by Aconitase to Form Isocitrate

Citrate is a poor substrate for oxidation

So aconitase isomerizes citrate to yield isocitrate which has asecondary-OH, which can be oxidized

Note the stereochemistry of the reaction: aconitase removes thepro-R H of the pro-R arm of citrate

Aconitase uses an iron-sulfur cluster - see Figure 19.8

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Figure 19.8 The aconitase reaction converts citrate to cis-aconitate

and then to isocitrate. Aconitase is stereospecific and removes the

pro-R hydrogen from the pro-R arm of citrate.


Figure 19.8 The active

site of aconitase. The

iron-sulfur cluster

(pink) is coordinated

by cysteines (orange)

and isocitrate

(purple).

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Fluoroacetate Blocks the TCA Cycle

Fluoroacetate is an extremely poisonous agent that blocks the TCA

cycle in vivo, although it has no apparent effect on any of the isolated

TCA cycle enzymes.

The action of aconitase has been traced to aconitase

Aconitase is inhibited by fluorocitrate, which is formed from

fluoracetate in two steps, as shown here.

Isocitrate Dehydrogenase Catalyzes the First Oxidative

Decarboxylation in the Cycle

Oxidative decarboxylation of isocitrate yields -ketoglutarate

This is classic NAD+ chemistry (hydride removal) followed by adecarboxylation

Isocitrate dehydrogenase is a link to the electron transport pathwaybecause it makes NADH

The mechanism is typical of NAD+-dependent enzymes (see Figure19.10)

The reaction involves (first) oxidation of the C-2 alcohol of isocitrate

to form oxalosuccinate, then a -decarboxylation reaction thatexpels the central carboxyl group as CO2

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Isocitrate Dehydrogenase Catalyzes the First Oxidative

Decarboxylation in the Cycle

Figure 19.10 The isocitrate

dehydrogenase reaction.

Hydride removal by NAD+ is

followed by a

-decarboxylation reaction that

expels the central carboxyl

group as CO2

-Ketoglutarate Dehydrogenase Catalyzes the SecondOxidative Decarboxylation of the TCA Cycle

A second oxidative decarboxylation

This enzyme is nearly identical to pyruvate dehydrogenase -structurally and mechanistically

Five coenzymes used - TPP, CoASH, lipoic acid, NAD+, and FAD

You know the mechanism if you remember pyruvatedehydrogenase

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-Ketoglutarate Dehydrogenase Catalyzes the Second OxidativeDecarboxylation of the TCA Cycle

Like pyruvate dehydrogenase, -ketoglutarate dehydrogenase is a

multienzyme complex consisting of-ketoglutaratedehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl

dehydrogenase. The complex uses five different coenzymes.

19.4 How Is Oxaloacetate Regenerated to Complete the

TCA Cycle?

Succinyl-CoA Synthetase Catalyzes

a Substrate-Level Phosphorylation

A nucleoside triphosphate is made

This is possible because succinyl-CoA is a high-energy

intermediate

Its hydrolysis (the hydrolysis of a CoA ester) drives the

phosphorylation of GDP to produce GTP

The mechanism (Figure 19.11) involves a phosphohistidine

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Succinate Dehydrogenase Is FAD-Dependent

An FAD-dependent oxidation of a single bond to a double bond The mechanism involves hydride removal by FAD and a

deprotonation

This enzyme is actually part of the electron transport pathwayin the inner mitochondrial membrane

The electrons transferred from succinate to FAD (to formFADH2) are passed directly to ubiquinone (UQ) in the electrontransport pathway

The Succinate Dehydrogenase Reaction

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Fumarase Catalyzes the trans-Hydration of Fumarate to

Form L-Malate

Hydration occurs across the newly formed double bond

Hydration involves trans-addition of the elements of water

across the double bond

Possible mechanisms are shown in Figure 19.13

The actual mechanism is not known for certain

The Fumarase Reaction

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The Malate Dehydrogenase Reaction

Steric Preferences in NAD+-Dependent Dehydrogenases

The dehydrogenases that require nicotinamidecoenzymes are stereospecific, and they transferhydride to either the pro-R or the pro-S positionsselectively

What accounts for this specificity? The enzymesinvolved are asymmetric structures.

The nicotinamide coenzyme (and substrate) fit into theactive site in only one way

The hydride transfers can only be accomplished to orfrom one side or the other of the NAD+ molecule

Dehydrogenases (and other enzymes too) are alsostereospecific with respect to the substrates as well

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The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA

Cycle

Figure 19.15 The fate of the carbon atoms of acetate in successive

TCA cycles.

The Carbon Atoms of Acetyl-CoA Have Different Fates in the

TCA Cycle

Figure 19.15

The fate of the

carbon atoms

of acetate in

successive TCAcycles.

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19.7 Can the TCA Cycle Provide Intermediates for

Biosynthesis?

The TCA cycle provides several of these

-Ketoglutarate is transaminated to make glutamate, which can beused to make purine nucleotides, as well as Arg and Pro

Succinyl-CoA can be used to make porphyrins

Fumarate and oxaloacetate can be used to make several amino acidsand also pyrimidine nucleotides

Note that mitochondrial citrate can be exported to be a cytoplasmicsource of acetyl-CoA and oxaloacetate

The TCA Cycle Can Provide Intermediates For Biosynthesis

Figure 19.16 TheTCA cycle

provides

intermediates for

biosynthesis.

Amino acids are

highlighted in

orange.

All 20 common

amino acids can

be made from

metabolites

derived from the

TCA cycle.

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19.8 What Are the Anaplerotic, or Filling Up, Reactions?

Pyruvate carboxylase - converts pyruvate to oxaloacetate

This is the most important anaplerotic reaction

PEP carboxylase - converts PEP to oxaloacetate

Malic enzyme converts pyruvate into malate

PEP carboxykinase - could have been an anaplerotic reaction, but itgoes the wrong way

CO2 binds weakly to PEP carboxykinase, but oxaloacetate bindstightly, so the reaction goes spontaneously in the oppositedirection.


Figure 19.17 Pyruvate carboxylase (shown here), and also

phosphoenolpyruvate (PEP) carboxylase, and malic enzymecatalyze anaplerotic reactions, replenishing TCA cycle

intermediates.

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19.8 What Are the Anaplerotic, orFilling Up, Reactions?

Figure 19.17 Pyruvate carboxylase , phosphoenolpyruvate (PEP)

carboxylase (shown here), and malic enzyme catalyze anaplerotic

reactions, replenishing TCA cycle intermediates.


Figure 19.17 Pyruvate carboxylase, and also phosphoenolpyruvate

(PEP) carboxylase and malic enzyme (shown here) catalyze anaplerotic

reactions, replenishing TCA cycle intermediates.

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Is PEP Carboxykinase an Anaplerotic Reaction?

This reaction *might* be an anaplerotic reaction, except for the

fact that CO2 binds weakly to PEP carboxykinase, and

oxaloacetate binds very tightly. As a result, the enzyme favors

formation of PEP from oxaloacetate.

Thus the reaction operates in the wrong direction to be an

anaplerotic reaction.

Anaplerosis Plays a Critical Role in Insulin Secretion

The pancreas releases insulin in response to an increase of

blood glucose

What cellular processes mediate this response?

It was long accepted that ATP produced by catabolism

activated K+ channels in the plasma membrane of -cells in

the pancreas

However, recent research has shown that anaplerotic

enzymes feed alternative pathways that produce cytosolic

signal molecules that also support insulin secretion

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The Reductive TCA Cycle

The TCA cycle running backward could assimilate CO2 This may have been the first metabolic pathway

This reductive TCA cycle occurs in certain extant archaea andbacteria, where it serves all their carbon needs

Energy to drive it? Maybe reaction of FeS with H2S to form FeS2(iron pyrite)

Iron pyrite, which was plentiful in ancient times, is an ancientversion ofiron-sulfur clusters found in many enzymes today!

The Reductive TCA Cycle

A reductive,

reversed TCA

cycle

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19.9 How Is the TCA Cycle Regulated?

As in TCA, 3 reactions are the key sites

Citrate synthase - ATP, NADH and succinyl-CoA inhibit

Isocitrate dehydrogenase - ATP inhibits, ADP and NAD+ activate

-Ketoglutarate dehydrogenase - NADH and succinyl-CoA inhibit,AMP activates

Also note pyruvate dehydrogenase:

ATP, NADH, acetyl-CoA inhibit

NAD+, CoA activate

Figure 19.18

Regulation of the TCA cycle.

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Pyruvate Dehydrogenase is Regulated by

Phosphorylation/Dephosphorylation

Figure 19.19 Regulation of

the pyruvate dehydrogenase

reaction.

Phosphorylation inactivates;

Dephosphorylation

activates.

19.10 Can Any Organisms Use Acetate as Their Sole

Carbon Source?

The Glyoxylate Cycle

Acetate-based growth - net synthesis of carbohydrates and otherintermediates from acetate - is not possible with TCA

The glyoxylate cycle offers a solution for plants and some bacteriaand algae

The CO2-evolving steps are bypassed and an extra acetate is utilized

Isocitrate lyase and malate synthase are the short-circuitingenzymes

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19.10 Can Any Organisms Use Acetate as Their Sole

Carbon Source?

Figure 19.20 The glyoxylate

cycle. The first two steps are

identical to TCA reactions.

Isocitrate lyase and malate

synthase short-circuit the TCA

cycle, forming malate and

succinate from isocitrate and

another acetyl-CoA.

More on the Glyoxylate Cycle

Isocitrate lyase produces glyoxylate and succinate

Malate synthase does a Claisen condensation of acetyl-CoA andthe aldehyde group of glyoxylate - classic CoA chemistry!

The glyoxylate cycle helps plants grow in the dark! Seeds are arich source of acetate (from fatty acids). Until the nascent plantsees the sun (and begins photosynthesis), it can grow using theglyoxylate cycle

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Isocitrate Lyase Short-Circuits the TCA Cycle by Producing

Glyoxylate and Succinate

Figure 19.21 The isocitrate lyase reaction. Isocitrate lyase catalyzes

an aldol cleavage and is similar to the aldolase reaction in glycolysis.

Glyoxysomes Must Borrow Three Reactions from

Mitochondria

Glyoxysomes do not contain all the enzymes needed to run

the glyoxylate cycle

Succinate dehydrogenase, fumarase, and malate

dehydrogenase are absent

Glyoxysomes borrow these three reactions from

mitochondria, so that they can convert succinate to

oxaloacetate

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Glyoxysomes Must Borrow Three Reactions from

Mitochondria

Figure 19.20

Glyoxysomes lack three

of the enzymes needed

to run the glyoxylate

cycle. Succinate

dehydrogenase,

fumarase, and malate

dehydrogenase are all

borrowed from

mitochondria.

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