Lecture Note 9

8/14/2019 Lecture Note 9

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catabolism degradation of nutrients to generate energy andstarting materials

anabolismbiosynthesis of biomolecules from starting

materials

metabolites substrates,intermediates and products of metabolism

Metabolism


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STAGE

I- Complexto Building

Blocks

II- BuildingBlocks to

Acetyl CoA

Stages of Catabolism

III-Oxidationof Acetyl

CoA

Each arrow represents acatabolic pathway.

Catabolic pathways

tend to converge.


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oxidativephosphorylation

LIPIDSPOLYSACCHARIDES

PROTEINS

fattyacids amino acids

acetyl-CoA

FADH 2 and NADH(reducing power)O 2

tricarboxylicacid (TCA)

cycle

pyruvate

glucose

ATP

ATP

Glycolysis


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Coenzyme A

performs a vital role by transporting acetylgroups from one substrate to another

the key to this action is the reactivethioester bond in the acetyl form of CoA

the thioester bond is stable enough that itcan survive inside the cell, but unstableenough that acetyl-CoA can readilytransfer the acetyl group to another molecule


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S C

O

C H3

H 2CH 2 CO

PhosphorylatedADP

PantothenicAcid

NH

AcylGroup

Mercaptoethylamine

CoEnzyme A


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Glycolysis

Glucose

Pyruvate Lactate

10 stepsNo O 2

3 regulated steps

Glycolysis takes place in the cytosol of cells.

Glucose enters the Glycolysis pathway byconversion to glucose-6-phosphate .


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2 Pyruvate + 2 ATP + 2 NADH + H 2O

Glucose + 2 ADP + 2 phosphate + 2 NAD +

Glycolysis


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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 (geneclusters)


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Reaction of pyruvate dehydrogenase complex (PDC)

Irreversible

acetyl-CoA cannot be converted back to pyruvate;

hence fat cannot be converted to carbohydrate


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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. Usuallyrequire NAD+ or FAD as co-factors/co-substrates)

Pathway Enzymes


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Mutase: group transfer enzyme. Common use is to movephosphates 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 anenzyme that combines two things to make a newcompound. (i.e. citrate synthase, succinyl CoA

synthetase).ATPase: Hydrolyses ATP to ADP and Pi. This reactionruns in reverse in FoF1 ATPase to generate ATP usingthe free energy of the proton gradient.

Pathway Enzymes


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Mg++

interacts with negatively charged phosphateoxygen atoms, providing charge compensation &promoting a favorable conformation of ATP at theactive site of the Hexokinase enzyme.

N

NN

N

NH 2

O

OHOH

HH

H

CH 2

H

OPOPOPO

O

O

O

O O

O

adenine

ribose

ATP

adenosine triphosphate


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The reaction catalyzed by Hexokinase is highly

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

The phosphate ester formed in glucose-6-phosphatehas a lower G of hydrolysis.

H O

OH

H

OHH

OH

CH 2OH

H

OH

H H O

OH

H

OHH

OH

CH 2OPO 32

H

OH

H

23

4

5

6

1 1

6

5

4

3 2

ATP ADP

Mg2+

glucose glucose-6-phosphateHexokinase


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Induced fit:

Binding of glucose to Hexokinase promotes a largeconformational change by stabilizing an alternativeconformation in which:

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

water is excluded from the active site. This preventsthe enzyme from catalyzing ATP hydrolysis.

glucose

Hexokinase


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Phosphofructokinase catalyzes:

fructose-6-P + ATP fructose-1,6-bisP + ADPThis highly spontaneous reaction.The Phosphofructokinase reaction is the rate-limitingstep of Glycolysis.

The enzyme is highly regulated .


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Pyruvate Kinase catalyzes:phosphoenolpyruvate + ADP pyruvate + ATP

C

C

CH 3

O O

O2

3

1ADP ATPC

C

CH 2

O O

OPO 32

2

3

1 C

C

CH 2

O O

OH2

3

1

phosphoenolpyruvate enolpyruvate pyruvate

Pyruvate Kinase


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C

C

CH 2OPO 32

H O

H OH

C

C

CH 2OPO 32

O OPO 32

H OH+P i

+ H +NAD+ NADH 1

2

3

2

3

1

glyceraldehyde- 1,3-bisphospho-3-phosphate glycerate

Glyceraldehyde-3-phosphateDehydrogenase

Glyceraldehyde-3-phosphate Dehydrogenase catalyzes: glyceraldehyde-3-P + NAD + + P i

1,3-bisphosphoglycerate + NADH + H+


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A cysteine thiol at the active site of Glyceraldehyde-

3-phosphate Dehydrogenase has a role in catalysis.The aldehyde of glyceraldehyde-3-phosphate reactswith the cysteine thiol to form a thiohemiacetal intermediate.

H3N+

C COO

CH 2

SH

H

cysteine

C

C

CH 2OPO 32

H O

H OH

1

2

3

glyceraldehyde-3- phosphate


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The high energy acyl thioester is attacked by P i to

yield the acyl phosphate ( ~P ) product.

CH CH 2OPO 32

OHEnz-Cys SH

Enz-Cys S CH CH CH 2OPO 32

OHOH

Enz-Cys S C CH CH 2OPO 32

OHO

HC

NAD+

NADH

Enz-Cys SH

P i

C CH CH 2OPO 32

OHO

O 3PO2

O

glyceraldehyde-3- phosphate

1,3-bisphosphoglycerate

thiohemiacetalintermediate

acyl-thioester intermediate

Oxidation to acarboxylic acid(in a ~ thioester )

occurs, as NAD + is reduced toNADH .


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

N

R

H

CNH2

O

N

R

CNH2

OH

H

+

2e

+ H+

AD + NADH


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CC

CH 3

O

O

O

CHC

CH 3

O

OH

O

NADH +

H

+

NAD+

Lactate Dehydrogenase

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.


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

The mechanism involves acid/base catalysis, with ringopening, isomerization via an enediolate intermediate ,and then ring closure.

H O

OH

H

OHH

OH

CH 2OPO 32

H

OH

H

1

6

5

4

3 2

CH 2OPO 32

OH

CH 2OH

H

OH H

H HO

O6

5

4 3

2

1

glucose-6-phosphate fructose-6-phosphatePhosphoglucose Isomerase


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Enolase catalyzes:2-phosphoglycerate phosphoenolpyruvate + H 2O

This dehydration reaction is Mg ++-dependent .

2 Mg ++ ions interact with oxygen atoms of the substratecarboxyl 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.

C

CCH 2OH

O O

H OPO 32

C

CCH 2OH

O O

OPO 32

C

CCH 2

O O

OPO 32

OH

2

3

1

2

3

1

H+

2-phosphoglycerate enolate intermediate phosphoenolpyruvate

Enolase


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C

C

CH 2OH

O O

H O PO 32

2

3

1C

C

CH 2OPO 32

O O

H OH2

3

1

3-phosphoglycerate 2-phosphoglycerate

Phosphoglycerate Mutase

Phosphoglycerate Mutase catalyzes:


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

Ph h l M


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C

C

CH 2OH

O O

H O PO 32

2

3

1C

C

CH 2OPO 32

O O

H OH2

3

1


Phosphoglycerate Mutase

C

C

CH 2OPO 32

O O

H OPO 322

3

1

2,3-bisphosphoglycerate

An active site histidine side-chain participates in P i transfer, bydonating & accepting phosphate.

The process involves a2,3-bisphosphate intermediate.

View an animation of the

Phosphoglycerate Mutase reaction.

H3N+

C COO

CH 2

CHN

HC NH

CH

H

+

histidine

2
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/glycolysis.htmhttp://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/glycolysis.htm


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Aldolase catalyzes:

fructose-1,6-bisphosphate dihydroxyacetone-P + glyceraldehyde-3-P

The reaction is an aldol cleavage , the reverse of an aldolcondensation. C atoms are renumbered in products of

Aldolase.

6

5

4

3

2

1CH 2OPO 32

C

C

C

C

CH 2OPO 32

O

HO H

H OH

H OH

3

2

1

CH 2OPO 32

C

CH 2OH

O

C

C

CH 2OPO 32

H O

H OH+

1

2

3

fructose-1,6- bisphosphate

Aldolase

dihydroxyacetone glyceraldehyde-3- phosphate phosphate

Triosephosphate Isomerase


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A lysine residue at the active site functions in catalysis.The keto group of fructose-1,6-bisphosphate reacts withthe -amino group of the active site lysine, to form aprotonated Schiff base intermediate.Cleavage of the bond between C3 & C4 follows.

CH 2OPO 3

2

C

CH

C

C

CH 2OPO 32

NH

HO

H OH

H OH

(CH 2)4 Enzyme

6

5

4

3

2

1

+

Schiff base intermediate of Aldolase reaction

H3N+ C COO

CH 2

CH 2

CH 2

CH 2

NH3

H

+

lysine

2


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Triose Phosphate Isomerase (TIM) catalyzes:dihydroxyacetone-P glyceraldehyde-3-P

Glycolysis continues from glyceraldehyde-3-P. TIM's K eq favors dihydroxyacetone-P. Removal of glyceraldehyde-3-P

by a subsequent spontaneous reaction allows throughput.

6

5

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2

1CH 2OPO 32

C

C

C

C

CH 2OPO 32

O

HO H

H OH

H OH

32

1

CH 2OPO 32

C

CH 2OH

O

C

C

CH 2OPO 32

H O

H OH+

1

2

3

fructose-1,6- bisphosphate

ldolase

dihydroxyacetone glyceraldehyde-3- phosphate phosphate



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The ketose/aldose conversion involves acid/basecatalysis, 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 2OPO 32

O

C

C

CH 2OPO 32

H O

H OH

C

C

CH 2OPO 32

H OH

OH

H

H OH H+

H+

H+

H+

dihydroxyacetone enediol glyceraldehyde- phosphate intermediate 3-phosphate



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Hexokinase

Phosphofructokinase

glucoseGlycolysis ATP

ADPglucose -6-phosphate

Phosphoglucose Isomerase

fructose -6-phosphate

ATPADP

fructose -1,6-bisphosphate

Aldolase

Glyceraldehyde-3-phosphate + Dihydroxyacetone phosphate Triosephosphate

IsomeraseGlycolysis continued


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Experimental approaches to study

metabolism Sequence of reaction pathway

Mechanistic analysis, metabolic pathwayinhibition

Regulation of pathway mechanism


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

Glucose-6-phosphate inhibits by competition at theactive site, as well as by allosteric interactions at aseparate site on the enzyme.

H O

OH

H

OHH

OH

CH 2OH

H

OH

H H O

OH

H

OHH

OH

CH 2OPO 32

H

OH

H

23

4

5

6

1 1

6

5

4

3 2

ATP ADP

Mg2+

glucose glucose-6-phosphate

Hexokinase


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Cells trap glucose by phosphorylating it, preventing exiton glucose carriers.

Product inhibition of Hexokinase ensures that cells willnot continue to accumulate glucose from the blood, if [glucose-6-phosphate] within the cell is ample.

H O

OH

H

OHH

OH

CH 2OH

H

OH

H H O

OH

H

OHH

OH

CH 2OPO 32

H

OH

H

23

4

5

6

1 1

6

5

4

3 2

ATP ADP

Mg2+

glucose glucose-6-phosphate

Hexokinase


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

Glucokinase is not subject to product inhibition byglucose-6-phosphate.

Liver will take up & phosphorylate glucose even when

liver [glucose-6-phosphate] is high.Liver Glucokinase is subject to inhibition by glucokinaseregulatory protein (GKRP ).

The ratio of Glucokinase to GKRP changes in differentmetabolic states, providing a mechanism for modulatingglucose phosphorylation.


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Glucokinase , with its high K M for glucose, allows the liver to store glucose as glycogen when blood [glucose] ishigh.

Glycogen Glucose

Hexokinase or GlucokinaseGlucose-6-Pase

Glucose-1-P Glucose-6-P Glucose + P i Glycolysis

PathwayPyruvate

Glucose metabolism in liver.


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Glucose-6-phosphatase catalyzes hydrolytic release of P i

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, bothfound 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 + P i Glycolysis Pathway

PyruvateGlucose metabolism in liver.


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Phosphofructokinase is usually the rate-limiting step of theGlycolysis pathway.Phosphofructokinase is allosterically inhibited by ATP .

At low concentration, the substrate ATP binds only atthe active site.At high concentration, ATP binds also at a low-affinityregulatory site, promoting the tense conformation.


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


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Inhibition of the Glycolysis enzyme Phosphofructokinasewhen [ATP] is high prevents breakdown of glucose in apathway whose main role is to make ATP.

It is more useful to the cell to store glucose as glycogenwhen ATP is plentiful.

Glycogen Glucose

Hexokinase or Glucokinase

Glucose-6-Pase Glucose-1-P Glucose-6-P Glucose + P i

Glycolysis Pathway

PyruvateGlucose metabolism in liver.

Lecture Note 9

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