Lecture Note 9

Metabolism catabolism – degradation of nutrients to generate energy and starting materials anabolism – biosynthesis of biomolecules from starting materials metabolites – substrates,intermediates and products of metabolism

Stages of Catabolism STAGE

I­ Complex  to Building  Blocks

Catabolic pathways tend to converge. Each arrow represents a catabolic pathway.

II­ Building  Blocks to  Acetyl CoA III­  Oxidation  of Acetyl  CoA

LIPIDS

POLYSACCHARIDES PROTEINS glucose

fatty acids

amino acids Glycolysis ATP pyruvate acetyl-CoA

O2

FADH2 and NADH (reducing power) oxidative phosphorylation ATP

tricarboxylic acid (TCA) cycle

Coenzyme A • performs a vital role by transporting acetyl groups from one substrate to another • the key to this action is the reactive thioester bond in the acetyl form of CoA • the thioester bond is stable enough that it can survive inside the cell, but unstable enough that acetyl-CoA can readily transfer the acetyl group to another molecule

O N H

O

Phosphorylated ADP

Pantothenic Acid

CoEnzyme A

H2C

H2 C

Mercaptoethylamine

S

C C H3

Acyl Group

Glycolysis Glycolysis takes place in the cytosol of cells. Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate. Glucose 3 regulated steps

10 steps Pyruvate

No O2

Lactate

Glycolysis

Glucose + 2 ADP + 2 phosphate + 2 NAD+

2 Pyruvate + 2 ATP + 2 NADH + H2O

• • • •

Metabolic pathways are irrereversible Every pathway has a first committed step All metabolic pathways are regulated Metabolic pathways in eukaryotic cells occur in specific cellular locations (gene clusters)

Reaction of pyruvate dehydrogenase complex (PDC)

Irreversible acetyl-CoA cannot be converted back to pyruvate; hence “fat cannot be converted to carbohydrate”

Pathway Enzymes Kinase: transfers a phosphate group from ATP (i.e. hexokinase, galactose kinase, pyruvate kinase) Isomerase: converts one isomer to another (i.e. phosphoglucoisomerase, triose phosphate isomerase) Aldolase: catalyzes aldol condensation(i.e. aldolase, functions in reverse in glycolysis) Dehydrogenase: removes hydrogens by oxidation. Usually require NAD+ or FAD as co-factors/co-substrates)

Pathway Enzymes Mutase: group transfer enzyme. Common use is to move phosphates to different positions on sugars (i.e. phosphoglycerate mutase, glucose-1-P mutase). Enolase: converts C=C group to alcohol. No change in oxidation state. Synthase: (also known as synthetase). Usually an enzyme that combines two things to make a new compound. (i.e. citrate synthase, succinyl CoA synthetase). ATPase: Hydrolyses ATP to ADP and Pi. This reaction runs in reverse in FoF1 ATPase to generate ATP using the free energy of the proton gradient.

6 CH2OH 5

H 4

OH

O

H OH

H 2

3

H

OH

glucose

6 CH OPO 2− 2 3 5 O

ATP ADP H H 1

OH

Mg2+

4

OH

Hexokinase

H OH

H 2

3

H

H 1

OH

OH

glucose-6-phosphate

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

N

adenosine triphosphate O −

O

O

P O

O −

P O

N

O O −

P O

O

CH2

−

H

N

N

adenine

O

H

H

OH

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 CH2OH 5

H 4

OH

O

H OH

H 2

3

H

OH

glucose

6 CH OPO 2− 2 3

ATP ADP H H 1

OH

Mg2+

5 4

OH

O

H OH 3

H 2

H 1

OH

Hexokinase H OH 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 ∆G of hydrolysis.

glucose

Hexokinase Induced fit: Binding of glucose to Hexokinase promotes a large conformational change by stabilizing an alternative conformation in which:  the C6 hydroxyl of the bound glucose is close to the terminal phosphate of ATP, promoting catalysis.  water is excluded from the active site. This prevents the enzyme from catalyzing ATP hydrolysis.

Phosphofructokinase catalyzes: fructose-6-P + ATP  fructose-1,6-bisP + ADP This highly spontaneous reaction. The Phosphofructokinase reaction is the rate-limiting step of Glycolysis. The enzyme is highly regulated.

Pyruvate Kinase O−

O C 1 C 2

ADP ATP

OPO32−

3 CH2

phosphoenolpyruvate

O−

O C

C

1

C

2

O−

O 1

OH

3 CH2

enolpyruvate

C

2

O

3 CH3

pyruvate

Pyruvate Kinase catalyzes: phosphoenolpyruvate + ADP  pyruvate + ATP

Glyceraldehyde-3-phosphate Dehydrogenase H

O

NAD+

1C

H

2

C

OH

+ Pi

2− CH OPO 2 3 3

glyceraldehyde3-phosphate

OPO32− + H+ O NADH 1C H

C

2

OH

2− CH OPO 2 3 3

1,3-bisphosphoglycerate

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

H

H H3N+

C

COO−

CH2 SH

cysteine

O

1C

H 2 C OH 2− 3 CH2OPO3

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

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

Enz-Cys

O

OH

HC

CH

SH

S

OH

OH

CH

CH

CH2OPO32−

glyceraldehyde-3phosphate CH2OPO32−

thiohemiacetal intermediate

NAD+ NADH

Enz-Cys

S

O

OH

C

CH

CH2OPO32−

acyl-thioester intermediate

Pi

Enz-Cys

SH

2−

O3PO

O

OH

C

CH

CH2OPO32−

1,3-bisphosphoglycerate

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

H

O C

+ N

H

H

C

NH2 −

O

+

2e + H

NH2

N

R

R

NAD+

NADH

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

Lactate Dehydrogenase O−

O C C

NADH + H+ NAD+

O

O−

O C HC

OH

CH3

CH3

pyruvate

lactate

Lactate is also a significant energy source for neurons in the brain. 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.

6 CH OPO 2− 2 3 5 O

H 4

OH

H OH 3

H

H 2

OH

H 1

OH

6 CH OPO 2− 2 3

1 CH2OH

O

5

H

H 4

OH

HO

2

3 OH

H

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

Enolase −

O

O C

1

H 2 C OPO32− 3 CH2OH

H+ − O

−

O C C

OH−

O−

O 1

OPO32−

CH2OH

C

2C

OPO32−

3 CH2

2-phosphoglycerate enolate intermediate phosphoenolpyruvate

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.

Phosphoglycerate Mutase O−

O C

1

O−

O C

1

H 2C OH 2− CH OPO 2 3 3

H 2C OPO32− 3 CH2OH

3-phosphoglycerate

2-phosphoglycerate

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

Phosphoglycerate Mutase O−

O C 1

H 2C OH 2− 3 CH2OPO3

3-phosphoglycerate

histidine

O−

O

H

C 1 H 2C OPO3 3 CH2OH

2−

2-phosphoglycerate

An active site histidine side-chain participates in Pi transfer, by donating & accepting phosphate. The process involves a 2,3-bisphosphate intermediate.

H3N+

COO−

C CH2 C

HN

CH

HC

NH +

O−

O C

1

H 2C OPO32− 2− 3 CH2OPO3

2,3-bisphosphoglycerate View an animation of the Phosphoglycerate Mutase reaction.

1CH2OPO3 2C

O

HO 3C H 4C

H

H

2−

Aldolase

H

2− CH OPO 2 3 3

1C

OH

2C

OH

1CH2OH

2− CH OPO 2 3 6

dihydroxyacetone phosphate

5

C

fructose-1,6bisphosphate

O

O

+

H 2C OH 2− 3 CH2OPO3

glyceraldehyde-3phosphate

Triosephosphate Isomerase

Aldolase catalyzes: fructose-1,6-bisphosphate  dihydroxyacetone-P + glyceraldehyde-3-P The reaction is an aldol cleavage, the reverse of an aldol condensation. C atoms are renumbered in products of Aldolase.

lysine

2− CH OPO 2 3 1

H +

H3N

C CH2 CH2 CH2 CH2 +

NH3

COO

2C

−

HO H H

3

CH C

OH

C

OH

4 5

NH (CH2)4 +

Enzyme

2− CH OPO 2 3 6

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 ε-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

HO 3C H 4C

H

H

2−

Aldolase

H

2− CH OPO 2 3 3

1C

OH

2C

OH

1CH2OH

2− CH OPO 2 3 6

dihydroxyacetone phosphate

5

C

fructose-1,6bisphosphate

O

O

+

H 2C OH 2− CH OPO 3 2 3

glyceraldehyde-3phosphate

Triosephosphate Isomerase

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

OH

C

O

+

H H

CH2OPO32−

dihydroxyacetone phosphate

+

H

OH C C

+

H H

OH

CH2OPO32−

enediol intermediate

+

H

O C

H

C

OH

CH2OPO32−

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.

Glycolysis

glucose ATP

Hexokinase

ADP glucose-6-phosphate

Phosphoglucose Isomerase fructose -6-phosphate ATP

Phosphofructokinase

ADP fructose-1,6-bisphosphate Aldolase

Glyceraldehyde-3-phosphate + Dihydroxyacetone phosphate Triosephosphate Isomerase Glycolysis continued

Experimental approaches to study metabolism • Sequence of reaction pathway • Mechanistic analysis, metabolic pathway inhibition • Regulation of pathway mechanism

6 CH2OH 5

H 4

OH

O

H OH

H 2

3

H

OH

glucose

6 CH OPO 2− 2 3 5 O

ATP ADP H H 1

OH

Mg2+

4

OH

H OH 3

H 2

H 1

OH

Hexokinase H OH glucose-6-phosphate

Hexokinase is inhibited by its product glucose-6phosphate. Glucose-6-phosphate inhibits by competition at the active site, as well as by allosteric interactions at a separate site on the enzyme.

6 CH2OH 5

H 4

OH

O

H OH

H 2

3

H

OH

glucose

6 CH OPO 2− 2 3

ATP ADP H H 1

OH

Mg2+

5 4

OH

O

H OH 3

H 1

H 2

OH

Hexokinase H OH glucose-6-phosphate

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.

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.

Glycogen

Glucose-1-P

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

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

Glycogen

Glucose-1-P

Glucose Hexokinase or Glucokinase Glucose-6-Pase 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].

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.

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.