Biochemistry of Metabolism

Glycolysis

H
4

6 CH OPO 2 2 3 5 O

H
1

H OH H
2 3

OH H 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 CH2OH

6 CH OPO 2 2 3 5

H H
2 1 4

O H
1

ATP ADP H H O H
2

H OH OH OH
3

Mg2+ OH H OH

H OH

OH OH

Hexokinase

glucose

glucose-6-phosphate

1. Hexokinase catalyzes: Glucose + ATP glucose-6-P + ADP

The reaction involves nucleophilic attack of the ! hydro"yl # of glucose on P of the ter$inal phosphate of ATP.

ATP binds to the enzy$e as a co$ple" with Mg++.

NH2

ATP
N N O O O H H OH O H H OH O P O P O CH2 O N N

adenosine triphosphate

adenine

ribose

Mg++ interacts with negatively charged phosphate o"ygen ato$s% providing charge co$pensation & pro$oting a favorable confor$ation of ATP at the active site of the 'e"okinase enzy$e.

6 CH2OH

6 CH OPO 2 2 3 5

H H
2 1 4

O H
1

ATP ADP H H O H
2

H OH OH OH
3

Mg2+

H OH OH

OH OH

glucose

Hexokinase H OH glucose-6-phosphate

The reaction catalyzed by 'e"okinase is highly spontaneous.

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

The phosphate ester for$ed in glucose*!* phosphate has a lower G of hydrolysis.

6 CH2OH

6 CH OPO 2 2 3 5

#n uce $it%
H
4

O H
1

ATP ADP H H O H
2 1 4

H OH H
2

OH
3 3

OH OH H

Mg

2+

H OH

OH OH

OH

Hexokinase

Glucose &in ing to 'e"okinase stabilizes a con$o!'ation in which:

glucose

glucose-6-phosphate

glucose

the C6 hy !oxyl of the bound glucose is close to the ter$inal phosphate of ATP% pro$oting catalysis.

Hexokinase

"ate! is exclu e fro$ the active site. This prevents the enzy$e fro$ catalyzing ATP hydrolysis% rather than transfer of phosphate to glucose.

glucose

Hexokinase

It is a co''on 'oti$ for an enzy$e active site to be located at an interface between protein do$ains that are connected by a fle"ible hinge region.

The st!uctu!al $lexi&ility allows access to the active site% while per$itting precise positioning of active site residues% and in so$e cases e"clusion of water% as substrate binding pro$otes a particular confor$ation.

+pecificity in 'e"okinase

'e"okinase has to discri$inate between the nor$al substrate% glucose% and water. The enzy$e has a $olecular weight of ,--%--- and contains two do$ains in which the active site resides at the cleft. .ater has access to the active% site% and can co$pete with the nor$al substrate to hydrolyze ATP into A/P and inorganic phosphate. 0pon binding of a sugar% however% the 1aws of the two lobes of the enzy$e collapse to for$ a properly oriented active site% with a$ino acids positioned to facilitate phosphoryl transfer. This allows the enzy$e to discri$inate against water by a factor of ,-!.

Hexokinase conformational change

The active site pocket changes shape upon binding glucose Only then can ATP transfer its phosphoryl group to the C6 carbon, yielding lu!6!P " A#P

Hexokinase conformational change

H O HO
3 OH 2

6 CH OPO 2 2 3 5 O

H
1 5

6 CH OPO 2 2 3 1CH2OH

H OH H
2

H
4

OH OH OH H

OH H

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

(. Phosphoglucose #so'e!ase catalyzes: glucose-6-P (aldose) $!uctose-6-P (ketose)

The $echanis$ involves acid2base catalysis% with ring opening% iso$erization via an ene iolate inte!'e iate% and then ring closure. A si$ilar reaction catalyzed by Triosephosphate Iso$erase will be presented in detail.

Phosphofructokinase
1CH2OH 6 CH OPO 2 2 3

6 CH OPO 2 2 3

O ATP ADP
2 5

O HO
3 OH 2

1CH2OPO32

5 3 OH 4

H Mg2+ H OH H

HO

H H

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.

1CH2OPO3

2C

O H
1C

H
2C

Aldolase
O

2 CH OPO 2 3 3

HO 3C H 4C

OH

H
1CH2OH

OH

H 2C OH 2 CH OPO 3 2 3

2 CH OPO 2 3 6

fructose-1,6bisphosphate

dihydroxyacetone glyceraldehyde-3phosphate phosphate

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
1CH2OPO3

H
2C

H 3N

COO

NH (CH2)4 + OH OH

Enzyme

HO
3 4 5

CH

CH2

H H
2 CH OPO 2 3 6

CH2

CH2

CH2

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 -amino group of the active site lysine, to form a protonated Schiff base intermediate.

Cleavage of the bond between C3 & C4 follows.

1CH2OPO3

2C

O H
1C

H
2C

Aldolase
O

2 CH OPO 2 3 3

HO 3C H 4C

OH

H
1CH2OH

OH

H 2C OH 2 CH OPO 3 2 3

2 CH OPO 2 3 6

fructose-1,6bisphosphate

dihydroxyacetone glyceraldehyde-3phosphate phosphate

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 H C OH CH2OPO32 C CH2OPO32 OH H H
+ + + +

H H OH O H

OH

CH2OPO32

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 O O C CH2OPO32 O C CH2OPO32

HC

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 barrel, or TIM barrel.

In an barrel there are 8 parallel -strands surrounded by 8 -helices.

TIM

Short loops connect alternating -strands & -helices.

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 -strands & loops connecting these to -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.

Glyceraldehyde-3-phosphate Dehydrogenase
O OH
2

1C

2 CH OPO 2 3 3

OPO32 + H+ O NAD+ NADH 1C + Pi H C OH

2 CH OPO 2 3 3

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
O NAD+ + Pi H
2 CH OPO 2 3 3

H OH
2

1C

OPO32 + H+ O NADH 1C C OH

2 CH OPO 2 3 3

glyceraldehyde3-phosphate

1,3-bisphosphoglycerate

3"ergonic o"idation of the aldehyde in glyceraldehyde* 4*phosphate% to a carbo"ylic acid% drives for$ation of an acyl phosphate% a 5high energy5 bond (~P).

This is the only step in Glycolysis in which )AD+ is reduced to 6A/'.

H
1C

H
COO

H3N+

CH2

H 2 C OH 2 3 CH2OPO3

SH

cysteine

glyceraldehyde-3phosphate

A cysteine thiol at the active site of Glyceraldehyde*4*phosphate /ehydrogenase has a role in catalysis.

The aldehyde of glyceraldehyde*4*phosphate reacts with the cysteine thiol to for$ a thiohe'iacetal inter$ediate.

Enz-Cys SH HC OH Enz-Cys S CH CH NAD+ NADH O Enz-Cys S Pi O Enz-Cys SH

2

O CH OH CH2OPO32 CH2OPO32

OH

glyceraldehyde-3phosphate thiohemiacetal intermediate

OH CH CH2OPO32

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

OH O3PO C CH

acyl-thioester intermediate
CH2OPO32

1,3-bisphosphoglycerate

The 7high energy8 acyl thioester is attacked by Pi to yield the acyl phosphate (~P) product.

H O H C NH2 NH2
+

+ N

2e + H
R

NAD+ NADH

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

Phosphoglycerate Kinase
OPO32 ADP ATP O
1

O C

1C

H 2C OH 2 3 CH2OPO3 Mg
2+

H 2C OH 2 3 CH2OPO3

1,3-bisphosphoglycerate

3-phosphoglycerate

7. Phosphoglycerate Kinase catalyzes: 1,3-bisphosphoglycerate + ADP 3-phosphoglycerate + ATP

This phosphate transfer is reversible (low G), since one ~P bond is cleaved & another synthesized.

The enzyme undergoes substrate-induced conformational change similar to that of Hexokinase.

Phosphoglycerate Mutase
O O
1 1

O C H 2C OPO32 3 CH2OH C

H 2C OH 2 3 CH2OPO3

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
2

O
H H3N+ C CH2 C HN HC
+

O H 2C OPO3 3 CH2OH
COO

histidine

C 1

H 2C OH 2 CH OPO 2 3 3

3-phosphoglycerate

2-phosphoglycerate

CH NH

An active site histidine side-chain participates in Pi transfer, by donating & accepting phosphate.

O
1

O C H 2C OPO32 2 3 CH2OPO3

The process involves a 2,3-bisphosphate intermediate.

2,3-bisphosphoglycerate

Enolase
H+ O O O
1 2C 3 CH2

O C C CH2OH OPO32 OPO32 C

OH O

H 2 C OPO32 3 CH2OH

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
1 2 3 CH3

O ADP ATP C O C O C 2
3 CH2

OPO32

phosphoenolpyruvate

pyruvate

10. Pyruvate Kinase catalyzes: phosphoenolpyruvate + ADP

pyruvate + ATP

Pyruvate Kinase
O ADP ATP
1 2 3 CH2 3 CH3

O C
1

O O C O C C
2

O OH

C 1

C 2

OPO32

3 CH2

phosphoenolpyruvate

enolpyruvate

pyruvate

This phosphate transfer from PEP to ADP is spontaneous.

PEP has a larger G 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 Phosphoglucose Isomerase Hexokinase

Glycolysis

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

Glycolysis continued.

Recall that there are 2 GAP per glucose.

Glycolysis

*alance sheet for ~P bonds of ATP:

2 'ow $any ATP ~P bonds e"pended9 ::::::::

'ow $any ~P bonds of ATP produced9 (;e$e$ber there are two 4 frag$ents fro$ 4 glucose.) ::::::::

6et production of ~P bonds of ATP per glucose: :::::::: 2

*alance sheet for ~P bonds of ATP: < ATP e"pended = ATP produced (< fro$ each of two 4 frag$ents fro$ glucose) 6et production of ( ~P bonds of ATP per glucose. Glycolysis * total pathway% o$itting '>: glucose + ( )AD+ + ( ADP + ( Pi ( py!u+ate + ( )ADH + ( ATP In ae!o&ic o!ganis's: py!u+ate produced in Glycolysis is o"idized to #< via ?rebs ycle )ADH produced in Glycolysis & ?rebs ycle is reo"idized via the respiratory chain% with production of $uch additional ATP.

Glyceraldehyde-3-phosphate Dehydrogenase
H
1C

O NAD+ + Pi H
2

Fermentation:
H
2 CH OPO 2 3 3

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

OH

Anaerobic organisms lack a respiratory chain.

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 CH3 CH3 O HC OH NADH + H+ NAD+ C O O

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 CH3 CH3 O HC OH NADH + H+ NAD+ C O O O

pyruvate

lactate

Skeletal muscles ferment glucose to lactate during exercise.

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 CH3 CH3 O HC OH NADH + H+ NAD+ C O O O

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.

Pyruvate Decarboxylase
CO2 H O C H CH3 C NADH + H+ NAD+ H OH CH3

Alcohol Dehydrogenase

CH3

pyruvate

acetaldehyde

ethanol

+o$e anaerobic organis$s $etabolize pyruvate to ethanol% which is e"creted as a waste product.

)ADH is converted to )AD+ in the reaction catalyzed by Alcohol /ehydrogenase.

Glycolysis% o$itting '>: glucose + ( )AD+ + ( ADP + ( Pi ( py!u+ate + ( )ADH + ( ATP

,e!'entation% fro$ glucose to lactate: glucose + ( ADP + ( Pi ( lactate + ( ATP

Anae!o&ic cata&olis' of glucose yields only < 7high energy8 bonds of ATP.

Glycolysis 3nzy$e2;eaction *<-.B *<C.< ><.< *,.= *,C.< *<D.B ><<.E *D.B >C.B negativ
e

G o@ G kA2$ol kA2$ol

'e"okinase Phosphoglucose Iso$erase Phosphofructokinase Aldolase Triosephosphate Iso$erase

*,!.C *,., Glyceraldehyde*4*P /ehydrogenase & Phosphoglycerate ?inase Phosphoglycerate Futase >=.C *-.! 3nolase *4.< *<.= rd *Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3 Edition, John Wiley & Sons, New York, p. 613. Pyruvate ?inase *<4.- *,4.B

,lux through the Glycolysis pathway is !egulate by control of 4 enzy$es that catalyze spontaneous reactions: Hexokinase% Phospho$!uctokinase & Py!u+ate -inase.

.ocal cont!ol of $etabolis$ involves regulatory effects of varied concentrations of pathway su&st!ates or inte!'e iates% to benefit the cell.

Glo&al cont!ol is for the benefit of the whole organis$% & often involves ho!'one-acti+ate signal casca es. .i+e! cells have $a1or roles in $etabolis$% including $aintaining blood levels various of nutrients such as glucose. Thus global control especially involves liver. +o$e aspects of global control by hor$one*activated signal cascades will be discussed later.

6 CH2OH

6 CH OPO 2 2 3 5

H H
2 1 4

O H
1

ATP ADP H H O H
2

H OH OH OH
3

Mg2+ OH

H OH

OH OH

glucose

Hexokinase H OH glucose-6-phosphate

Hexokinase is inhi&ite by p!o uct glucose-6phosphate: by co'petition at the acti+e site by alloste!ic interaction at a sepa!ate enzy$e site.

ells t!ap glucose by phospho!ylating it% preventing e"it on glucose carriers.

P!o uct inhi&ition of 'e"okinase ensures that cells will not continue to accu$ulate glucose fro$ the blood% if Gglucose*!* phosphateH within the cell is a$ple.

6 CH2OH

6 CH OPO 2 2 3 5

H
4

O H
1

ATP ADP H H O H
2 1 4

H OH H
2

OH
3 3

OH OH

Mg2+

H OH

OH

Glucokinase is a variant of 'e"okinase found in li+e!.

H OH

glucose

Hexokinase H OH glucose-6-phosphate

Glucokinase has a high -M for glucose. It is acti+e only at high /glucose0.

#ne effect of insulin% a hor$one produced when blood glucose is high% is acti+ation in liver of t!ansc!iption of the gene that encodes the Glucokinase enzy$e.

Glucokinase is not su&1ect to p!o uct inhi&ition by glucose*!*phosphate. Iiver will take up & phosphorylate glucose even when liver Gglucose*!*phosphateH is high.

Glucokinase is sub1ect to inhi&ition by glucokinase !egulato!y p!otein (G-2P).

The ratio of Glucokinase to G?;P in liver changes in different $etabolic states% providing a $echanis$ for $odulating glucose phosphorylation.

Glycogen

Glucose-1-P

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

Glucokinase, with high KM for glucose, allows liver to store glucose as glycogen in the fed state when blood [glucose] is high.

Pyruvate Glucose metabolism in liver.

Glucose-6-phosphatase catalyzes hydrolytic release of Pi fro$ glucose*!*P. Thus glucose is !elease fro$ the liver to the blood as needed to $aintain blood GglucoseH.

The enzy$es Glucokinase & Glucose*!*phosphatase% both found in li+e! but not in $ost other body cells% allow the liver to control blood GglucoseH.

Pyruvate Kinase
O C 1
1 2

O ADP ATP C C O
3 CH3

C 2
3 CH2

Py!u+ate -inase% the last step Glycolysis% is cont!olle in li+e! partly by $odulation of the a'ount of en4y'e.
OPO32

phosphoenolpyruvate

pyruvate

High /glucose0 within liver cells causes a transcription factor ca!&ohy !ate !esponsi+e ele'ent &in ing p!otein (Ch23*P) to be transferred into the nucleus% where it activates t!ansc!iption of the gene for Pyruvate ?inase.

This facilitates converting excess glucose to py!u+ate% which is $etabolized to acetyl-CoA% the $ain precursor for synthesis of $atty aci s% for long ter$ energy storage.

Phosphofructokinase
1CH2OH 6 CH OPO 2 2 3

6 CH OPO 2 2 3

O ATP ADP
2 5

O HO
3 OH 2

1CH2OPO32

5 3 OH 4

H Mg2+ H OH H

HO

H H

OH

fructose-6-phosphate

fructose-1,6-bisphosphate

Phospho$!uctokinase is usually the !ate-li'iting step of the Glycolysis pathway.

Phosphofructokinase is alloste!ically inhi&ite &y ATP.

At lo" concentration% the substrate ATP binds only at the acti+e site.

At high concentration% ATP binds also at a low*affinity !egulato!y site% pro$oting the tense confor$ation.

60

50

low [ATP]

40

30

PFK Activity

20

high [ATP]

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

The tense confor$ation of PJ?% at high /ATP0% has lower affinity for the other substrate% fructose*!*P. 5ig'oi al dependence of reaction rate on Gfructose*!*PH is seen. AMP% present at significant levels only when there is e"tensive 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 enzy$e Phosphofructokinase when GATPH is high prevents breakdown of glucose in a pathway whose $ain role is to $ake ATP.

It is $ore useful to the cell to store glucose as glycogen when ATP is plentiful.