G l i k o l i z

.

Glikoliz , hücrenin sitozolunda meydana gelir.

Glukoz, glukoz-6-fosfata dönüşerek glikoliz yoluna girer.

Başlangıçta, ATP’nin iki ~P bağının kırılmasına bağlı olarak enerji girişi olur.

H O

OH

H

OHH

OH

CH2OPO32

H

OH

H

1

6

5

4

3 2

glucose-6-phosphate

H O

O H

H

O HH

O H

CH 2O H

H

O H

H H O

O H

H

O HH

O H

CH 2O PO 32

H

O H

H

23

4

5

6

1 1

6

5

4

3 2

A T P A D P

M g 2+

glucose g lucose -6 -phosphate

H ex ok inase

1. Hekzokinz aşağıdaki reaksiyonu katalizler Glukoz + ATP glukoz-6-P + ADP

Reaksiyon, glukozun C6 hidroksil O’nin ATP’nin terminal P’e nukleofilik atak yapmasını içerir.

ATP ,enzime Mg++ le kompleks oluşturarak bağlanır..

Mg++ negatif yüklü fosfat esteri ile etkileşir bu şekilde ATP’nin hekzokinaz enziminin aktif merkezi için uygun yük konformasyonunu sağlar.

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OPOPOP O

O

O O

O O

O

adenine

ribose

ATP adenosine triphosphate

Heksokinaz la katalize edilen reaksiyon yüksek derecede spontandır..

ATP’nin fosfoanhidrid (~P) bağı kırılır.

Glukoz-6-fosfatta oluşan fosfat ester bağı düşük G’ya sahiptir

H O

O H

H

O HH

O H

CH 2O H

H

O H

H H O

O H

H

O HH

O H

CH 2O PO 32

H

O H

H

23

4

5

6

1 1

6

5

4

3 2

A T P A D P

M g 2+

glu co se g lu co se -6 -p h osp h ate

H ex ok in ase

Bu da glukozun C6 OH’nin, ATP’nin terminal Pi yakınlaşmasını sağlar ve aktif bölgeden suyun çıkarılmasına neden olur.. Bu olay da ATP’nin hidrolizini önler ve P transferine olanak sağlar.

glucose

Hexokinase

Glukozun hekzokinaza bağlanması önemli yapısal değişime neden olur.

2. Fosfogluko Izomeraz reaksiyonu: glukoz-6-P (aldoz) fruktoz-6-P (ketoz)

Mekanizma, asit/baz katalizini içerir, halka açılması, enediolat arametaboliti ile izomerizasyon, ve halka kapanması gözlenir. Triozfosfat Izomeraz ile katalize edilen benzer bir reaksiyon daha detalı olarak gösterilecektir.

H O

O H

H

O HH

O H

CH 2 O PO 32

H

O H

H

1

6

5

4

3 2

CH 2 O PO 32

O H

CH 2 O H

H

O H H

H HO

O6

5

4 3

2

1

g lu c o s e - 6 - p h o s p h a te f r u c to s e - 6 - p h o s p h a te P h o s p h o g lu c o s e Is o m e r a s e

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.

CH 2 O PO 32

O H

CH 2 O H

H

O H H

H HO

O6

5

4 3

2

1 CH 2 O PO 32

O H

CH 2 O PO 32

H

O H H

H HO

O6

5

4 3

2

1

A T P A D P

M g 2 +

f r u c t o s e - 6 - p h o s p h a t e f r u c t o s e - 1 , 6 - b i s p h o s p h a t e

P h o s p h o f r u c t o k i n a s e

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.

6

5

4

3

2

1 CH 2 O PO 32

C

C

C

C

CH 2 O PO 32

O

HO H

H O H

H O H

3

2

1

CH 2 O PO 32

C

CH 2 O H

O

C

C

CH 2 O PO 32

H O

H O H+

1

2

3

f ru c to s e -1 ,6 - b is p h o s p h a te

A ld o la s e

d ih y d ro x y a c e to n e g ly c e ra ld e h y d e -3 - p h o s p h a te p h o s p h a te

T rio s e p h o s p h a te Is o m e ra s e

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.

CH2OPO3

2

C

CH

C

C

CH2OPO32

NH

HO

H OH

H OH

(CH2)4 Enzyme

6

5

4

3

2

1

+

Schiff base intermediate of Aldolase reaction

H3N+ C COO

CH2

CH2

CH2

CH2

NH3

H

lysine

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.

6

5

4

3

2

1 CH 2 O PO 32

C

C

C

C

CH 2 O PO 32

O

HO H

H O H

H O H

3

2

1

CH 2 O PO 32

C

CH 2 O H

O

C

C

CH 2 O PO 32

H O

H O H+

1

2

3

f ru c to s e -1 ,6 - b is p h o s p h a te

A ld o la s e

d ih y d ro x y a c e to n e g ly c e ra ld e h y d e -3 - p h o s p h a te p h o s p h a te

T rio s e p h o s p h a te Is o m e ra s e

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.

C

C

CH 2 O PO 32

O

C

C

CH 2 O PO 32

H O

H O H

C

C

CH 2 O PO 32

H O H

O H

H

H O H H + H + H + H +

d i h y d r o x y a c e t o n e e n e d i o l g l y c e r a l d e h y d e - p h o s p h a t e i n t e r m e d i a t e 3 - p h o s p h a t e

T r i o s e p h o s p h a t e I s o m e r a s e

C

CH 2 O PO 32

O O

C

CH 2 O PO 32

HC O

O H

p r o p o s e d e n e d i o l a t e

i n t e r m e d i a t e

p h o s p h o g l y c o l a t e t r a n s i t i o n s t a t e

a n a l o g

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.

TIM

Triosephosphate Isomerase structure is an barrel, or TIM barrel.

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

Short loops connect alternating -strands & -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 -strands & loops connecting these to -helices.

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.

TIM

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.

C

CH 2 O PO 32

O O

C

CH 2 O PO 32

HC O

O H

p r o p o s e d e n e d i o l a t e

i n t e r m e d i a t e

p h o s p h o g l y c o l a t e t r a n s i t i o n s t a t e

a n a l o g

C

C

CH 2 O PO 32

H O

H O H

C

C

CH 2 O PO 32

O O PO 32

H O H+ P i

+ H +

N A D + N A D H 1

2

3

2

3

1

g l y c e r a l d e h y d e - 1 , 3 - b i s p h o s p h o - 3 - p h o s p h a t e g l y c e r a t e

G l y c e r a l d e h y d e - 3 - p h o s p h a t e D e h y d r o g e n a s e

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

C

C

CH 2 O PO 32

H O

H O H

C

C

CH 2 O PO 32

O O PO 32

H O H+ P i

+ H +

N A D + N A D H 1

2

3

2

3

1

g l y c e r a l d e h y d e - 1 , 3 - b i s p h o s p h o - 3 - p h o s p h a t e g l y c e r a t e

G l y c e r a l d e h y d e - 3 - p h o s p h a t e D e h y d r o g e n a s e

Exergonic oxidation of the aldehyde in glyceraldehyde- 3-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.

A cysteine thiol at the active site of Glyceraldehyde-3-phosphate Dehydrogenase has a role in catalysis.

The aldehyde of glyceraldehyde-3-phosphate reacts with the cysteine thiol to form a thiohemiacetal intermediate.

H3N+ C COO

CH2

SH

H

cysteine

C

C

CH2OPO32

H O

H OH

1

2

3

glyceraldehyde-3- phosphate

The “high energy” acyl thioester is attacked by Pi to

yield the acyl phosphate (~P) product.

CH CH2OPO32

OHEnz-Cys SH

Enz-Cys S CH CH CH2OPO32

OHOH

Enz-Cys S C CH CH2OPO32

OHO

HC

NAD +

NADH

Enz-Cys SH

Pi

C CH CH2OPO32

OHO

O3PO2

O

glyceraldehyde-3-phosphate

1,3-bisphosphoglycerate

thiohemiacetal intermediate

acyl-thioester intermediate

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

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

N

R

H

CN H 2

O

N

R

CN H 2

OH H

+

2 e + H

+

N A D + N A D H

C

C

CH 2 O PO 32

O O PO 32

H O H

C

C

CH 2 O PO 32

O O

H O H

A D P A T P

1

22

3 3

1

M g 2+

1 , 3 - b i s p h o s p h o - 3 - p h o s p h o g l y c e r a t e g l y c e r a t e

P h o s p h o g l y c e r a t e K i n a s e

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

C

C

CH 2 O H

O O

H O PO 32

2

3

1C

C

CH 2 O PO 32

O O

H O H2

3

1

3 - p h o s p h o g l y c e r a t e 2 - p h o s p h o g l y c e r a t e

P h o s p h o g l y c e r a t e M u t a s e

8. Phosphoglycerate Mutase catalyzes: 3-phosphoglycerate 2-phosphoglycerate

Phosphate is shifted from the OH on C3 to the OH on C2.

C

C

CH 2 O H

O O

H O PO 32

2

3

1C

C

CH 2 O PO 32

O O

H O H2

3

1

3 - p h o s p h o g l y c e r a t e 2 - p h o s p h o g l y c e r a t e

P h o s p h o g l y c e r a t e M u t a s e

C

C

CH2OPO32

O O

H OPO32

2

3

1

2,3-bisphosphoglycerate

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

The process involves a 2,3-bisphosphate intermediate.View an animation of the Phosphoglycerate Mutase reaction.

H3N+ C COO

CH2

CHN

HC NH

CH

H

histidine

C

C

CH 2 O H

O O

H O PO 32

2

3

1C

C

CH 2

O O

O PO 32

2

3

1

+ H 2 O

2 - p h o s p h o g l y c e r a t e p h o s p h o e n o l p y r u v a t e

E n o l a s e

9. Enolase catalyzes

2-phosphoglycerate phosphoenolpyruvate + H2O

This Mg++-dependent dehydration reaction is inhibited by fluoride.

Fluorophosphate forms a complex with Mg++ at the active site.

10. Pyruvate Kinase catalyzes:

phosphoenolpyruvate + ADP pyruvate + ATP

This reaction 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.

C

C

CH 3

O O

O2

3

1A D P A T PC

C

CH 2

O O

O PO 32

2

3

1 C

C

CH 2

O O

O H2

3

1

p h o s p h o e n o l p y r u v a t e e n o l p y r u v a t e p y r u v a t e

P y r u v a t e K i n a s e

Hexokinase

Phosphofructokinase

glucose Glycolysis

ATP

ADP glucose-6-phosphate

Phosphoglucose Isomerase

fructose-6-phosphate

ATP

ADP fructose-1,6-bisphosphate

Aldolase

glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate

Triosephosphate Isomerase Glycolysis continued

Glyceraldehyde-3-phosphate Dehydrogenase

Phosphoglycerate Kinase

Enolase

Pyruvate Kinase

glyceraldehyde-3-phosphate

NAD+ + Pi

NADH + H+

1,3-bisphosphoglycerate

ADP

ATP

3-phosphoglycerate

Phosphoglycerate Mutase

2-phosphoglycerate H2O

phosphoenolpyruvate

ADP

ATP pyruvate

Glycolysis continued.

Recall that there are 2 GAP per glucose.

Glycolysis

Balance sheet for ~P bonds of ATP:

How many ATP ~P bonds expended? ________

How many ~P bonds of ATP produced? (Remember there are two 3C fragments from glucose.) ________

Net production of ~P bonds of ATP per glucose: ________

2

4

2

Glycolysis

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

Fermentation

Anaerobes lack a respiratory chain for reoxidizing NADH. They must reoxidize NADH through some other reaction.

NAD+ is needed for Glyceraldehyde-3-P Dehydrogenase of Glycolysis.

C

C

CH 2 O PO 32

H O

H O H

C

C

CH 2 O PO 32

O O PO 32

H O H+ P i

+ H +

N A D + N A D H 1

2

3

2

3

1

g l y c e r a l d e h y d e - 1 , 3 - b i s p h o s p h o - 3 - p h o s p h a t e g l y c e r a t e

G l y c e r a l d e h y d e - 3 - p h o s p h a t e D e h y d r o g e n a s e

C

C

CH 3

O

O

O

C

H C

CH 3

O

O H

ON A D H + H + N A D +

L a c t a t e D e h y d r o g e n a s e

p y r u v a t e l a c t a t e

Skeletal muscles function anaerobically in exercise, when aerobic metabolism cannot keep up with energy needs.

Pyruvate is converted to lactate, regenerating NAD+

needed for Glycolysis.

Glycolysis is the main source of ATP under anaerobic conditions.

C

C

CH 3

O

O

O

C

CH 3

O HC

CH 3

OH H

H

N A D H + H + N A D +CO 2

P y r u v a t e A l c o h o l D e c a r b o x y l a s e D e h y d r o g e n a s e

p y r u v a t e a c e t a l d e h y d e e t h a n o l

Fermentation

Some anaerobic organisms metabolize pyruvate to ethanol, which is excreted as a waste product.

The Alcohol Dehydrogenase reaction regenerates NAD+, needed for continuation of Glycolysis.

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

Anaerobes excrete the product of fermentation (e.g., lactate or ethanol). They derive only 2 ATP from glucose catabolism.

In aerobic organisms, pyruvate is instead oxidized further to CO2, via Krebs Cycle and oxidative phosphorylation, with production of additional ATP.

Glycolysis Enzyme/ReactionGo'

kJ/molG

kJ/mol

Hexokinase -20.9 -27.2

Phosphoglucose Isomerase +2.2 -1.4

Phosphofructokinase -17.2 -25.9

Aldolase +22.8 -5.9

Triosephosphate Isomerase +7.9 negative

Glyceraldehyde-3-P Dehydrogenase& Phosphoglycerate Kinase

-16.7 -1.1

Phosphoglycerate Mutase +4.7 -0.6

Enolase -3.2 -2.4

Pyruvate Kinase -23.0 -13.9*Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3rd Edition, John Wiley & Sons, New York, p. 613.

Three Glycolysis enzymes catalyze spontaneous reactions: Hexokinase, Phosphofructokinase & Pyruvate Kinase.

Control of these enzymes determines the rate of the Glycolysis pathway.

Local control involves dependence of enzyme-catalyzed reactions on concentrations of pathway substrates or intermediates within a cell.

Global control involves hormone-activated production of second messengers that regulate cellular reactions for the benefit of the organism as a whole.

Local control will be discussed here. Regulation by hormone-activated cAMP signal cascade will be discussed later.

Hexokinase is inhibited by its product glucose-6-phosphate.

Glucose-6-phosphate inhibits by competition at the active site, as well as by allosteric interactions at a separate site on the enzyme.

H O

O H

H

O HH

O H

CH 2O H

H

O H

H H O

O H

H

O HH

O H

CH 2O PO 32

H

O H

H

23

4

5

6

1 1

6

5

4

3 2

A T P A D P

M g 2+

glu co se g lu co se -6 -p h osp h ate

H ex ok in ase

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.

H O

O H

H

O HH

O H

CH 2O H

H

O H

H H O

O H

H

O HH

O H

CH 2O PO 32

H

O H

H

23

4

5

6

1 1

6

5

4

3 2

A T P A D P

M g 2+

glu co se g lu co se -6 -p h osp h ate

H ex ok in ase

Glucokinase, a variant of Hexokinase found in liver, has a high KM for glucose. It is active only at high [glucose].

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.

Liver Glucokinase is subject to inhibition by glucokinase regulatory protein (GKRP).

The ratio of Glucokinase to GKRP changes in different metabolic states, providing a mechanism for modulating glucose phosphorylation.

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

Glycogen Glucose

Hexokinase or Glucokinase

Glucose-6-Pase Glucose-1-P Glucose-6-P Glucose + Pi Glycolysis Pathway

Pyruvate Glucose metabolism in liver.

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].

Glycogen Glucose

Hexokinase or Glucokinase

Glucose-6-Pase Glucose-1-P Glucose-6-P Glucose + Pi Glycolysis Pathway

Pyruvate Glucose metabolism in liver.

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.

CH 2 O PO 32

O H

CH 2 O H

H

O H H

H HO

O6

5

4 3

2

1 CH 2 O PO 32

O H

CH 2 O PO 32

H

O H H

H HO

O6

5

4 3

2

1

A T P A D P

M g 2 +

f r u c t o s e - 6 - p h o s p h a t e f r u c t o s e - 1 , 6 - b i s p h o s p h a t e

P h o s p h o f r u c t o k i n a s e

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.

0

10

20

30

40

50

60

0 0.5 1 1.5 2[Fructose-6-phosphate] m M

PFK

Act

ivity

high [AT P]

low [A T P]

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.

Glycogen Glucose

Hexokinase or Glucokinase

Glucose-6-Pase Glucose-1-P Glucose-6-P Glucose + Pi Glycolysis Pathway

Pyruvate Glucose metabolism in liver.