课件-生物化学2

Lecture3 Citric

Acid

CycleTricarboxylic

acid

cycle(TCA

cycle

or

Krebs

cycle)Zhang

Mengjie:

cherry5

:1IntroductionProduction

of

Acetyl-CoAReactions

of

the

Citric

Acid

CycleRegulation

of

the

Citric

Acid

CycleThe

Glyoxylate

Cycle(乙醛酸循环）outline23The

tricarboxylic

acid

(TCA)

cycle

is

also

known

as

theKrebs

cycle

or

the

citric

acid

cycle.TCA

cycle

is

the

hub

of

intermediarymetabolism

serving

both

the

catabolic

andanabolic

process-----

Amphibolic

pathway1.

Introduction1953

Nobel

Laureate

in

Medicinefor

his

discovery

of

the

citric

acid

cycleBackground1900-1981Place

of

Birth:

Hildesheim,

GermanyResidence:

Great

BritainAffiliation:

Sheffield

UniversityHans

Krebs,

1900–19814HANS

ADOLF

KREBS5The

TCA

cycle

is

a

vital

step

in

the

aerobiccreation

of

ATP,

the

primary

fuel

for

cellularactivityTCA

Take

place－－Eukaryotes:

mitochondriaProkaryotes:

cytosol7内膜蛋白对应线虫的eat-38外膜融合蛋白对应线虫fzo-1It

isthe

second

ofthreesteps

in

cellularrespiration,occurring

betweenglycolysis

(the

breakdownof

glucose

into

pyruvate)

andoxidative

phosphorylation(the

creation

of

ATP)cellular

respiration

--Rather

than

being

reducedto

lactate,

ethanol,

or

some

other

fermentationproduct,

the

pyruvate

produced

by

glycolysis

isfurther

oxidized

to

H2O

and

CO2.11TCA

cycle

is

coupled

to

oxidative

phosphorylation122.

Production

of

Acetyl-CoA-----Pyruvate

OxidationA

Major

Entry

Route

for

Carbon

into

the

Citric

Acid

CyclePyruvate

Dehydrogenase

Complex酸脱氢酶系1314Pyruvate

Dehydrogenase

Complex

（PDC）

three

enzymes

(E1,

E2

andE3):Pyruvate

Dehydrogenase(E1)

酸脱氢酶Dihydrolipoyl

transacetylase(E2)

二氢硫辛酰转乙酰基酶二氢硫辛酰脱氢酶Dihydrolipoyl

dehydrogenase

(E3)five

coenzymes:Thiamine

Pyrophosphate

焦磷酸硫胺素,辅羧酶(TPP)…VB1Lipoic

Acid硫辛酸-lipoamide硫辛酰胺CoASH…泛酸NAD+…

VPP5.

FAD…

VB2is

the

coenzyme

form

of

VB1Dissociable

protonThe

reactive

carbon

atomPyruvate

Dehydrogenase

(E1)酸脱氢酶羟乙基TPP（HETPP)15①

The

TPP

carbanion(负碳离子)actsas

anucleophile(亲核),attacking

the

carbonylgroup

of

pyruvate.16②

Decarboxylation

produces

a

carbanionthat

is

stabilized

by

the

thiazolium(噻唑)ring.③

Protonation

to

form

hydroxyethyl

TPP④

release

of

acetaldehyde.⑤

A

proton

dissociates

to

regenerate

thecarbanion.羟乙基TPPLipoic

acid（硫辛酸）Lipoic

acid

is

found

in

pyruvate

dehydrogenase

and

α-ketoglutaratedehydrogenaseLipoic

acid

is

covalently

bound

with

ε-NH2

of

a

lysine

chainLipoic

acid

functions

to

couple

acyl-group

transfer

and

electron

transferDihydrolipoyl

transacetylase

(E2)二氢硫辛酰转乙酰基酶17Lipoic

acid（硫辛酸）G3P-DH

is

the

site

of

action

of

arsenate

(砷酸盐)19Coenzymes

A20Dihydrolipoyl

dehydrogenase

(E3)二氢硫辛酰脱氢酶The

favin

coenzymes—FAD/FADH221二氢硫辛酰脱氢酶22二氢硫辛酰转乙酰基酶The

core

(green)

consists

of

24

molecules

of

E2,

arranged

in

8

trimersThe

lipoyl

硫辛酰基(blue)

of

E2

reaches

outward

to

touch

the

active

sites

of

E1The

swinging

arm

on

E2

can

reach

the

active

sites

of

E3

subunits

(red).2324central

metabolic

pathway中心代谢途径generates

NADH

and

FADH2produces

GTP

via

substra evel

phosphorylationMany

metabolic

processes

use

intermediates

of

theTCA

in

their

pathways.3.

Reactions

of

TCA25cyclic

pathway"begin"

with

addition

of

acetyl-CoA

to

oxaloacetate

toform

citrate26The

TCA

Has

Eight

StepsSteps1,3,and

4

areessentially

irreversiblein

the

cellFour

stepsin

thisprocess

areoxidations,

energy

ofoxidation

----

NADHand

FADH2.①③Step5

may

be

either

ATP

or

GTP④Phase

1: Introduction

and

Loss

of

Two

Carbon

AtomsStep

1:

Introduction

of

Two

Carbon

Atoms

as

Acetyl-CoAStep

2:Isomerization（异构化）

of

Citrate（柠檬酸）Step

3:

Generation

of

CO2

by

an NAD+

Linked

DehydrogenaseStep

4:

Generation

of

a

Second

CO2

by

a

Multienzyme

ComplexPhase

2:

Regeneration

of

Oxaloacetate（草酰乙酸）Step

5:

A

Substra evel

PhosphorylationStep

6:

A

Flavin-Dependent

DehydrogenaseStep

7:Hydration

水合作用of

a

Carbon-Carbon

Double

BondStep

8:

A

Dehydrogenation

that

Regenerates

OxaloacetateThe

Citric

Acid

Cycle

Has

Eight

Steps28Step

1:

Introduction

of

Two

Carbon

Atoms

as

Acetyl-CoA29柠檬酸合酶citrate

synthaseTCA

限速酶The

large,

negative

standard

free-energy

change

of

the

citratesynthase

reaction

is

essential

to

the

operation

of

the

cyclePhase

1:

Introduction

and

Loss

of

Two

Carbon

Atoms30Structure

of

citratesynthase（二聚体）open

form

of

the

enzyme

aloneclosed

form

with

bound

oxaloacetate

and

a

stableacetyl-CoA

.og

ofThe

flexible of

each

subunit

undergoes

a

largeconformational

change

on

binding

oxaloacetatecreating

a

binding

site

for

acetyl-CoA.oxaloacetateacetyl-CoAStep

2:

Isomerization

of

Citrate顺乌头酸叔醇化合物Aconitase

乌头酸酶柠檬酸异柠檬酸化合物（可氧化）31The

iron-sulfur

center

acts

in

both

substrate

binding

and

catalysis.Three

Cys

residues

of

the

enzyme

bind

three

iron

atomsThe

fourth

iron

is

bound

to

one

of

the

carboxyl

groups

of

citrateand

also

interacts

noncovalently

with

-OH

group

of

citrate.A

basic

residue

(:B)

on

the

enzyme

helps

to

position

the

citrate

in

theactive

site.32Aconitase乌头酸酶Step

3:

Generation

of

CO2

byan NAD+

Linked

DehydrogenaseIsocitrate

dehydrogenase

异柠檬酸脱氢酶酮酸的形成可促进

C-C键的断裂，有利于脱羧作用的进行ADP草酰琥珀酸ATP，NADH

inhibit

the

activity

of

isocitrate

dehydrogenase异柠檬酸α-酮戊二酸3334Step

4Generation

of

a

Second

CO2

by

a

Multienzyme

Complexα-酮戊二酸α

-KG

脱氢酶系1.

α

-KG

dehydrogenase

(E1)Dihydrolipoamide

transacetylase

(E2)二氢硫辛酰转琥珀酰酶Dihydrolipoamide

dehydrogenase

(E3)二氢硫辛酰脱氢酶five

coenzymesThiamine

Pyrophosphate焦磷酸硫胺素,辅羧酶(TPP)Lipoic

Acid-lipoamide硫辛酰胺FADNAD+CoASH琥珀酰CoA3536Step

5:

Conversion

of

Succinyl-CoA

to

Succinate琥珀酰－CoA

酶底物水平磷酸化--------Only

one

in

TCAIn

animal:

GTPIn

plant

and

microorganism:

ATPPhase

2:Regeneration

of

Oxaloacetate（草酰乙酸）琥珀酰CoA琥珀酸Different

from

synthase①

a

phosphoryl

group

replaces

the

CoA

of37succinyl-CoA

bound

to

the

enzyme,forming

a

high-energy

acyl

phosphate.酰基磷酸②

the

succinyl

phosphate

donatesits

phosphoryl

group

to

a

Hisresidue

on

the

enzyme,forming

a

high-energyphosphohistidyl

enzyme③

the

phosphoryl

group

istransferred

from

the

Hisresidue

to

the

terminalphosphate

of

GDP

(or

ADP),forming

GTP

(or

ATP).38Step

6:

Oxidation

of

Succinate

to

Fumarate-琥珀酸脱氢酶succinate

dehydrogenase--A

Flavin-Dependent

DehydrogenaseH+H+琥珀酸39延胡索酸Only

enzyme

of

TCA

is

membrane-boundMalonate

(丙二酸),

og

of

succinate,

is

a

strongcompetitive

inhibitor.40Step

7:

Hydration

of

Fumarate

to

Malate41fumarase延胡索酸酶Fumarase-----highly

stereospecific延胡索酸L-苹果酸Step

8:

Oxidation

of

Malate

to

Oxaloacetate苹果酸脱氢酶Malatedehydrogenase42L-苹果酸草酰乙酸43For

each

acetyl-CoA

oxidizedby

the

citric

acid

cycle3

NADH1

FADH21

ATP

orGTPThe

Energy

of

Oxidations

in

the

Cycle4445Note:A

two-carbon

acetyl

group

entered

the

cycle

bycombining

with

oxaloacetate.Two

carbon

atoms

emerged

from

the

cycle

as

CO2

fromthe

oxidation

of

isocitrate

and

α-ketoglutarate.The

two

carbon

atoms

appearing

as

CO2

arenotthesame

two

carbons

that

entered

in

the

form

of

the

acetylgroup46Energetics

of

the

Citric

Acid

Cycle10

ATP30-32ATP4748Besides

acetyl-CoA，any

compound

that

gives

riseto

a

four-

or

five-carbon

intermediate

of

the

citric

acidcycle—can

be

oxidized

by

thecycleThe

TCA

is

amphibolic

(双重代谢的)serving

in

both

catabolism

andanabolism.The

TCA

cycle

provides

intermediates

forbiosynthesis

besides

generating

energyPorphyrins

and

HemeFatty

acid

biosynthesisPyrimidine

biosynthesisPurine

biosynthesisAmino

acidbiosynthesisAmino

acid

biosynthesis49Amino

acidbiosynthesisAnaplerotic

reactions

to

fill

up

TCA回补反应50Theplete

TCA

Cyclein

prokaryotes514.

Regulation

of

Pyruvate

Dehydrogenaseand

the

Citric

Acid

Cycleregulated

in

two

primary

wayscontrolling

the

entry

of

fuel

into

the

cyclecontrolling

key

reactions

within

the

cycle5253Pyruvate

dehydrogenase

---

major

regulatory

point

for

entry

of

materials

into

TCAThe

enzyme

is

regulated

allosterically

and

by

covalentmodification.Allosteric

Regulation（E2

&

E3）：E2

-

inhibited

by

acetyl-CoA,

activated

byCoA-SHE3

-inhibited

by

NADH,

activated

by

NAD+.ATP:

allosteric

inhibitor

of

the

complexAMP:

activator4

.1 Controlling

the

entry

of

fuel

into

the

cycleCovalent

Regulation

(E1)Regulation

by

the

level

of

NADH/NAD+

ratio55564

.2

Key

reactions

within

the

cycle:allosteric

regulation

&

Covalent

Regulationisocitrate

dehydrogenaseActivated

by

ADP,

inhibited

by

NADHPhosphorylation

of

one

serine

residue

in

the

enzyme

preventsbinding

of

isocitrateα-ketoglutarate

dehydrogenaseInhibited

by

succinyl-CoA

and

NADHPlantssome

bacteria

(including

E.

coli

and

yeast)converting

acetate（乙酸）

to

carbohydrate.isocitra yase

异柠檬酸裂合酶malate

synthase

苹果酸合酶uses

some

of

the

same

enzymes

as

the

citric

acid

cyclebut

no

decarboxylations（脱羧）

steps5.Glyoxylate

Cycle

乙醛酸循环57One

cycle

has

five

stepsLacks

CO2

liberating

reactionsConsumes

two

acetyl-CoAsand

one

oxaloacetateProduces

two

oxaloacetates(Succinate

produced

fromisocitrate

is

converted

to

anotheroxaloacetate)乙醛酸乙醛酸60Pathway

for

the

formation

of

glucose

fromnoncarbohydrate

precursors

in

plants,

bacteria

andyeast

(not

animals)Glyoxylate

cycle

leads

from

2-carbon

compoundsto

glucoseIn

animals,

acetyl

CoA

is

not

a

carbon