Biochemistry of Metabolism

Glycolysis

Copyright 1998-2007 by Joyce J. Diwan. All rights reserved.

H
4

6 CH OPO 2 2 3 5 O

H
1

H OH
3

H
2

OH

OH

OH

glucose-6-phosphate

Glycolysis takes place in the cytosol of cells. Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate.

Initially there is energy input corresponding to cleavage of two ~P bonds of ATP.

6 CH 2OH

H
4

O H
2

ATP ADP H H
1 4

6 CH OPO 2 2 3 5

O H
2

H
1

H OH
3

OH

OH

Mg2+ OH

H OH
3

OH

OH

Hexokinase

OH

glucose

glucose-6-phosphate

1. Hexokinase catalyzes: Glucose + ATP glucose-6-P + ADP The reaction involves nucleophilic attack of the C6 hydroxyl O of glucose on P of the terminal phosphate of ATP. ATP binds to the enzyme as a complex with Mg++.

NH2

ATP
adenosine triphosphate
O

O O

O O

N O

P O

P O

P O

CH2 H H OH

adenine
O H H OH

ribose

Mg++ interacts with negatively charged phosphate oxygen atoms, providing charge compensation & promoting a favorable conformation of ATP at the active site of the Hexokinase enzyme.

6 CH 2OH

H
4

O H
2

ATP ADP H H
1 4

6 CH OPO 2 2 3 5

O H
2

H
1

H OH
3

OH

OH

Mg2+ OH

H OH
3

OH

OH

Hexokinase

OH

glucose

glucose-6-phosphate

The reaction catalyzed by Hexokinase is highly spontaneous.

A phosphoanhydride bond of ATP (~P) is cleaved.

The phosphate ester formed in glucose-6-phosphate has a lower DG of hydrolysis.

6 CH 2OH

Induced fit:

H
4

O H
2

ATP ADP H H
1 4

6 CH OPO 2 2 3 5

O H
2

H
1

Glucose binding to Hexokinase stabilizes a conformation in which:

H OH
3

OH

OH

Mg

2+

H OH
3

OH

OH

OH

Hexokinase

OH

glucose

glucose-6-phosphate

glucose

the C6 hydroxyl of the bound glucose is close to the terminal phosphate of ATP, promoting catalysis.

Hexokinase

water is excluded from the active site. This prevents the enzyme from catalyzing ATP hydrolysis, rather than transfer of phosphate to glucose.

glucose

Hexokinase

It is a common motif for an enzyme active site to be located at an interface between protein domains that are connected by a flexible hinge region. The structural flexibility allows access to the active site, while permitting precise positioning of active site residues, and in some cases exclusion of water, as substrate binding promotes a particular conformation.

6 CH OPO 2 2 3

H
4

O H
2

H
1

6 CH OPO 2 2 3

H OH
3

1 CH 2OH

H
4

HO H

OH

OH

3 OH

OH

OH

Phosphoglucose Isomerase glucose-6-phosphate fructose-6-phosphate

2. Phosphoglucose Isomerase catalyzes: glucose-6-P (aldose) fructose-6-P (ketose)

The mechanism involves acid/base catalysis, with ring opening, isomerization via an enediolate intermediate, and then ring closure. A similar reaction catalyzed by Triosephosphate Isomerase will be presented in detail.

Phosphofructokinase
6 CH OPO 2 2 3

1CH2OH

6 CH OPO 2 2 3

ATP ADP HO H
2 5

1CH2OPO32

H
4

H
4

HO H

3 OH

Mg2+

3 OH

OH

OH

fructose-6-phosphate

fructose-1,6-bisphosphate

3. Phosphofructokinase catalyzes: fructose-6-P + ATP fructose-1,6-bisP + ADP This highly spontaneous reaction has a mechanism similar to that of Hexokinase. The Phosphofructokinase reaction is the rate-limiting step of Glycolysis. The enzyme is highly regulated, as will be discussed later.

1CH 2OPO3 2C

O H OH OH

HO 3C H 4C H
5

Aldolase

2 CH OPO 2 3 3

H
1C

2C

1CH 2OH

H 2C OH 2 3 CH 2OPO3

2 CH OPO 2 3 6

fructose-1,6bisphosphate

dihydroxyacetone phosphate

glyceraldehyde-3phosphate

Triosephosphate Isomerase

4. Aldolase catalyzes: fructose-1,6-bisphosphate dihydroxyacetone-P + glyceraldehyde-3-P

The reaction is an aldol cleavage, the reverse of an aldol condensation. Note that C atoms are renumbered in products of Aldolase.

lysine
H H 3N
+

1CH2OPO3

C CH2 CH2 CH2 CH2

COO

2C

NH (CH2)4 + OH OH

Enzyme

HO H H

CH C C

4 5

2 CH OPO 2 3 6

NH3

Schiff base intermediate of Aldolase reaction

A lysine residue at the active site functions in catalysis.

The keto group of fructose-1,6-bisphosphate reacts with the e-amino group of the active site lysine, to form a protonated Schiff base intermediate. Cleavage of the bond between C3 & C4 follows.

1CH 2OPO3 2C

O H OH OH

HO 3C H 4C H
5

Aldolase

2 CH OPO 2 3 3

H
1C

2C

1CH 2OH

H 2C OH 2 3 CH 2OPO3

2 CH OPO 2 3 6

fructose-1,6bisphosphate

dihydroxyacetone phosphate

glyceraldehyde-3phosphate

Triosephosphate Isomerase

5. Triose Phosphate Isomerase (TIM) catalyzes: dihydroxyacetone-P glyceraldehyde-3-P Glycolysis continues from glyceraldehyde-3-P. TIM's Keq favors dihydroxyacetone-P. Removal of glyceraldehyde-3-P by a subsequent spontaneous reaction allows throughput.

Triosephosphate Isomerase
H H C C OH O H H
+ +

H C C

OH OH

H H

H C H C

O OH

CH 2OPO32

CH 2OPO32

CH 2OPO32

dihydroxyacetone phosphate

enediol intermediate

glyceraldehyde3-phosphate

The ketose/aldose conversion involves acid/base catalysis, and is thought to proceed via an enediol intermediate, as with Phosphoglucose Isomerase. Active site Glu and His residues are thought to extract and donate protons during catalysis.

OH HC C CH 2OPO 32 O O C CH 2OPO 32 O

proposed enediolate intermediate

phosphoglycolate transition state analog

2-Phosphoglycolate is a transition state analog that binds tightly at the active site of Triose Phosphate Isomerase (TIM). This inhibitor of catalysis by TIM is similar in structure to the proposed enediolate intermediate.

TIM is judged a "perfect enzyme." Reaction rate is limited only by the rate that substrate collides with the enzyme.

Triosephosphate Isomerase structure is an ab barrel, or TIM barrel.

In an ab barrel there are 8 parallel b-strands surrounded by 8 a-helices. Short loops connect alternating b-strands & a-helices.
TIM

TIM barrels serve as scaffolds for active site residues in a diverse array of enzymes. Residues of the active site are always at the same end of the barrel, on C-terminal ends of b-strands & loops connecting these to a-helices.

TIM

There is debate whether the many different enzymes with TIM barrel structures are evolutionarily related. In spite of the structural similarities there is tremendous diversity in catalytic functions of these enzymes and little sequence homology.

OH HC C CH 2OPO 32 O O C CH 2OPO 32 O

TIM

proposed enediolate intermediate

phosphoglycolate transition state analog

Explore the structure of the Triosephosphate Isomerase (TIM) homodimer, with the transition state inhibitor 2-phosphoglycolate bound to one of the TIM monomers. Note the structure of the TIM barrel, and the loop that forms a lid that closes over the active site after binding of the substrate.

Glyceraldehyde-3-phosphate Dehydrogenase
H
1C

O C OH

NAD+ + Pi

OPO32 + H+ O NADH 1C H
2 CH OPO 2 3 3

2 CH OPO 2 3 3

OH

glyceraldehyde3-phosphate

1,3-bisphosphoglycerate

6. Glyceraldehyde-3-phosphate Dehydrogenase catalyzes: glyceraldehyde-3-P + NAD+ + Pi 1,3-bisphosphoglycerate + NADH + H+

Glyceraldehyde-3-phosphate Dehydrogenase
H
1C

O C OH

NAD+ + Pi

OPO32 + H+ O NADH 1C H
2 CH OPO 2 3 3

2 CH OPO 2 3 3

OH

glyceraldehyde3-phosphate

1,3-bisphosphoglycerate

Exergonic oxidation of the aldehyde in glyceraldehyde3-phosphate, to a carboxylic acid, drives formation of an acyl phosphate, a "high energy" bond (~P). This is the only step in Glycolysis in which NAD+ is reduced to NADH.

H H3N+ C CH2 SH COO

H
1C

H 2 C OH 2 3 CH2OPO3

cysteine

glyceraldehyde-3phosphate

A cysteine thiol at the active site of Glyceraldehyde3-phosphate Dehydrogenase has a role in catalysis. The aldehyde of glyceraldehyde-3-phosphate reacts with the cysteine thiol to form a thiohemiacetal intermediate.

Enz-Cys

SH

O HC OH

OH CH OH CH CH2OPO32

glyceraldehyde-3phosphate
CH2OPO32

Oxidation to a carboxylic acid (in a ~ thioester) occurs, as NAD+ is reduced to NADH.

Enz-Cys

S NAD+ NADH

CH

thiohemiacetal intermediate
O OH CH CH2OPO32

Enz-Cys

S Pi

acyl-thioester intermediate
O OH CH CH2OPO32
2

Enz-Cys

SH

O3PO

1,3-bisphosphoglycerate

The high energy acyl thioester is attacked by Pi to yield the acyl phosphate (~P) product.

O C NH2

O C NH2

+ N R

2e + H

N R

NAD+

NADH

Recall that NAD+ accepts 2 e plus one H+ (a hydride) in going to its reduced form.

Phosphoglycerate Kinase
O
1C

OPO32 ADP ATP O

1

O C

H 2C OH 2 CH OPO 2 3 3

Mg2+

H 2C OH 2 CH OPO 2 3 3

1,3-bisphosphoglycerate

3-phosphoglycerate

7. Phosphoglycerate Kinase catalyzes: 1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP This phosphate transfer is reversible (low DG), since one ~P bond is cleaved & another synthesized.
The enzyme undergoes substrate-induced conformational change similar to that of Hexokinase.

Phosphoglycerate Mutase
O
1

O C

O
1

O C

H 2C OH 2 CH OPO 2 3 3

H 2C OPO32 3 CH 2OH

3-phosphoglycerate

2-phosphoglycerate

8. Phosphoglycerate Mutase catalyzes: 3-phosphoglycerate 2-phosphoglycerate Phosphate is shifted from the OH on C3 to the OH on C2.

Phosphoglycerate Mutase
O C 1 H 2C OH 2 CH OPO 2 3 3 O O C 1 H 2C OPO3 3 CH 2OH O
2

histidine
H H3N+ C CH2 C HN HC

COO

3-phosphoglycerate

2-phosphoglycerate

CH NH

An active site histidine side-chain participates in Pi transfer, by donating & accepting phosphate. The process involves a 2,3-bisphosphate intermediate. View an animation of the Phosphoglycerate Mutase reaction.

O
1

O C

H 2C OPO32 2 CH OPO 2 3 3

2,3-bisphosphoglycerate

Enolase
O
1

O C

O C C

OH

O
1

O C OPO32

H 2 C OPO32 3 CH2OH

OPO32

2C

CH2OH

3 CH2

2-phosphoglycerate enolate intermediate phosphoenolpyruvate

9. Enolase catalyzes: 2-phosphoglycerate phosphoenolpyruvate + H2O

This dehydration reaction is Mg++-dependent.

2 Mg++ ions interact with oxygen atoms of the substrate carboxyl group at the active site.

The Mg++ ions help to stabilize the enolate anion intermediate that forms when a Lys extracts H+ from C #2.

Pyruvate Kinase
O C 1 C 2 O OPO32 ADP ATP O
1 2

O C C O

3 CH 2

3 CH 3

phosphoenolpyruvate

pyruvate

10. Pyruvate Kinase catalyzes: phosphoenolpyruvate + ADP pyruvate + ATP

Pyruvate Kinase
O C 1 C 2 O ADP ATP OPO32 O
1 2

O C C OH

O
1 2

O C C O

3 CH 2

3 CH 2

3 CH 3

phosphoenolpyruvate

enolpyruvate

pyruvate

This phosphate transfer from PEP to ADP is spontaneous.

PEP has a larger DG of phosphate hydrolysis than ATP. Removal of Pi from PEP yields an unstable enol, which spontaneously converts to the keto form of pyruvate. Required inorganic cations K+ and Mg++ bind to anionic residues at the active site of Pyruvate Kinase.

glucose ATP ADP glucose-6-phosphate Hexokinase

Glycolysis

Phosphoglucose Isomerase fructose-6-phosphate ATP Phosphofructokinase ADP fructose-1,6-bisphosphate Aldolase glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate Triosephosphate Isomerase Glycolysis continued

glyceraldehyde-3-phosphate NAD+ + Pi Glyceraldehyde-3-phosphate Dehydrogenase NADH + H+

Glycolysis continued. Recall that there are 2 GAP per glucose.

1,3-bisphosphoglycerate ADP Phosphoglycerate Kinase ATP 3-phosphoglycerate Phosphoglycerate Mutase 2-phosphoglycerate Enolase H2O phosphoenolpyruvate ADP Pyruvate Kinase ATP pyruvate

Glycolysis
Balance sheet for ~P bonds of ATP:
2 How many ATP ~P bonds expended? ________

How many ~P bonds of ATP produced? (Remember 4 there are two 3C fragments from glucose.) ________
Net production of ~P bonds of ATP per glucose: ________ 2

Balance sheet for ~P bonds of ATP: 2 ATP expended 4 ATP produced (2 from each of two 3C fragments from glucose) Net production of 2 ~P bonds of ATP per glucose. Glycolysis - total pathway, omitting H+: glucose + 2 NAD+ + 2 ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATP In aerobic organisms: pyruvate produced in Glycolysis is oxidized to CO2 via Krebs Cycle NADH produced in Glycolysis & Krebs Cycle is reoxidized via the respiratory chain, with production of much additional ATP.

Glyceraldehyde-3-phosphate Dehydrogenase
H O
1C

NAD+ + Pi

Fermentation: Anaerobic organisms lack a respiratory chain.

OPO32 + H+ O NADH 1C H
2 CH OPO 2 3 3

2 CH OPO 2 3 3

OH

OH

glyceraldehyde3-phosphate

1,3-bisphosphoglycerate

They must reoxidize NADH produced in Glycolysis through some other reaction, because NAD+ is needed for the Glyceraldehyde-3-phosphate Dehydrogenase reaction. Usually NADH is reoxidized as pyruvate is converted to a more reduced compound.
The complete pathway, including Glycolysis and the reoxidation of NADH, is called fermentation.

Lactate Dehydrogenase
O C C O O NADH + H+ NAD+ O C HC OH O

CH3

CH3

pyruvate

lactate

E.g., Lactate Dehydrogenase catalyzes reduction of the keto in pyruvate to a hydroxyl, yielding lactate, as NADH is oxidized to NAD+. Lactate, in addition to being an end-product of fermentation, serves as a mobile form of nutrient energy, & possibly as a signal molecule in mammalian organisms. Cell membranes contain carrier proteins that facilitate transport of lactate.

Lactate Dehydrogenase
O C C O O NADH + H+ NAD+ O C HC OH O

CH3

CH3

pyruvate

lactate

Skeletal muscles ferment glucose to lactate during exercise, when the exertion is brief and intense.

Lactate released to the blood may be taken up by other tissues, or by skeletal muscle after exercise, and converted via Lactate Dehydrogenase back to pyruvate, which may be oxidized in Krebs Cycle or (in liver) converted to back to glucose via gluconeogenesis

Lactate Dehydrogenase
O C C O O NADH + H+ NAD+ O C HC OH O

CH3

CH3

pyruvate

lactate

Lactate serves as a fuel source for cardiac muscle as well as brain neurons. Astrocytes, which surround and protect neurons in the brain, ferment glucose to lactate and release it.

Lactate taken up by adjacent neurons is converted to pyruvate that is oxidized via Krebs Cycle.

O C C

O O

Pyruvate Decarboxylase
CO 2 H C CH 3 O

Alcohol Dehydrogenase
NADH + H+ NAD+ H H C OH

CH 3

CH 3

pyruvate

acetaldehyde

ethanol

Some anaerobic organisms metabolize pyruvate to ethanol, which is excreted as a waste product. NADH is converted to NAD+ in the reaction catalyzed by Alcohol Dehydrogenase.

Glycolysis, omitting H+: glucose + 2 NAD+ + 2 ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATP Fermentation, from glucose to lactate: glucose + 2 ADP + 2 Pi 2 lactate + 2 ATP Anaerobic catabolism of glucose yields only 2 high energy bonds of ATP.

Glycolysis Enzyme/Reaction Hexokinase Phosphoglucose Isomerase Phosphofructokinase Aldolase Triosephosphate Isomerase Glyceraldehyde-3-P Dehydrogenase & Phosphoglycerate Kinase Phosphoglycerate Mutase Enolase Pyruvate Kinase

DGo' DG kJ/mol kJ/mol -20.9 -27.2 +2.2 -1.4 -17.2 -25.9 +22.8 -5.9 +7.9 negative -16.7 -1.1 +4.7 -3.2 -23.0 -0.6 -2.4 -13.9

*Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3rd Edition, John Wiley & Sons, New York, p. 613.

Flux through the Glycolysis pathway is regulated by control of 3 enzymes that catalyze spontaneous reactions: Hexokinase, Phosphofructokinase & Pyruvate Kinase.

Local control of metabolism involves regulatory effects of varied concentrations of pathway substrates or intermediates, to benefit the cell. Global control is for the benefit of the whole organism, & often involves hormone-activated signal cascades.
Liver cells have major roles in metabolism, including maintaining blood levels various of nutrients such as glucose. Thus global control especially involves liver. Some aspects of global control by hormone-activated signal cascades will be discussed later.

6 CH 2OH

H
4

O H
2

ATP ADP H H
1 4

6 CH OPO 2 2 3 5

O H
2

H
1

H OH
3

OH

OH

Mg2+ OH

H OH
3

OH

OH

Hexokinase

OH

glucose

glucose-6-phosphate

Hexokinase is inhibited by product glucose-6-phosphate: by competition at the active site by allosteric interaction at a separate enzyme site. Cells trap glucose by phosphorylating it, preventing exit on glucose carriers. Product inhibition of Hexokinase ensures that cells will not continue to accumulate glucose from the blood, if [glucose-6-phosphate] within the cell is ample.

6 CH 2OH

5 4

O H
2

ATP ADP H H
1 4

6 CH OPO 2 2 3 5

O H
2

H
1

Glucokinase is a variant of Hexokinase found in liver.

H OH
3

OH

OH

Mg2+ OH

H OH
3

OH

OH

Hexokinase

OH

glucose

glucose-6-phosphate

Glucokinase has a high KM for glucose. It is active only at high [glucose]. One effect of insulin, a hormone produced when blood glucose is high, is activation in liver of transcription of the gene that encodes the Glucokinase enzyme. Glucokinase is not subject to product inhibition by glucose-6-phosphate. Liver will take up & phosphorylate glucose even when liver [glucose-6-phosphate] is high.

 Glucokinase is subject to inhibition by glucokinase regulatory protein (GKRP). The ratio of Glucokinase to GKRP in liver changes in different metabolic states, providing a mechanism for modulating glucose phosphorylation.

Glycogen

Glucokinase, with high KM Glucose-1-P for glucose, allows liver to store glucose Pyruvate as glycogen in Glucose metabolism in liver. the fed state when blood [glucose] is high.

Glucose Hexokinase or Glucokinase Glucose-6-Pase Glucose-6-P Glucose + Pi Glycolysis Pathway

Glucose-6-phosphatase catalyzes hydrolytic release of Pi from glucose-6-P. Thus glucose is released from the liver to the blood as needed to maintain blood [glucose]. The enzymes Glucokinase & Glucose-6-phosphatase, both found in liver but not in most other body cells, allow the liver to control blood [glucose].

Pyruvate Kinase
O O Pyruvate Kinase, the ADP C last step Glycolysis, is 1 2 C OPO controlled in liver partly 3 2 by modulation of the 3 CH 2 amount of enzyme. phosphoenolpyruvate ATP O
1 2

O C C O

3 CH 3

pyruvate

High [glucose] within liver cells causes a transcription factor carbohydrate responsive element binding protein (ChREBP) to be transferred into the nucleus, where it activates transcription of the gene for Pyruvate Kinase.
This facilitates converting excess glucose to pyruvate, which is metabolized to acetyl-CoA, the main precursor for synthesis of fatty acids, for long term energy storage.

Phosphofructokinase
6 CH OPO 2 2 3

1CH2OH

6 CH OPO 2 2 3

ATP ADP HO H
2 5

1CH2OPO32

H
4

H
4

HO H

3 OH

Mg2+

3 OH

OH

OH

fructose-6-phosphate

fructose-1,6-bisphosphate

Phosphofructokinase is usually the rate-limiting step of the Glycolysis pathway.

Phosphofructokinase is allosterically inhibited by ATP.

At low concentration, the substrate ATP binds only at the active site.

At high concentration, ATP binds also at a low-affinity regulatory site, promoting the tense conformation.

60 50

low [ATP]

PFK Activity

40 30 20 10 0 0 0.5 1 1.5 [Fructose-6-phosphate] mM 2

high [ATP]

The tense conformation of PFK, at high [ATP], has lower affinity for the other substrate, fructose-6-P. Sigmoidal dependence of reaction rate on [fructose-6-P] is seen. AMP, present at significant levels only when there is extensive ATP hydrolysis, antagonizes effects of high ATP.

Glycogen

Glucose-1-P

Glucose Hexokinase or Glucokinase Glucose-6-Pase Glucose-6-P Glucose + Pi Glycolysis Pathway

Pyruvate Glucose metabolism in liver.

Inhibition of the Glycolysis enzyme Phosphofructokinase when [ATP] is high prevents breakdown of glucose in a pathway whose main role is to make ATP. It is more useful to the cell to store glucose as glycogen when ATP is plentiful.