Chapter 3 Chapter 3 Metabolisms of CarbohydratesMetabolisms of Carbohydrates

3. Aerobic catalysis of carbohydrate 3. Aerobic catalysis of carbohydrate and tricarboxylic acid cycleand tricarboxylic acid cycle 糖的有氧分解与三羧酸循环糖的有氧分解与三羧酸循环

3.1 A survey of introduction 3.1 A survey of introduction

概述概述

三羧酸循环（ 三羧酸循环（ TTririccarboxylic arboxylic aacid cid ccycle)ycle)

TCA Cycle TACTCA Cycle TAC Kreb’sKreb’s 循环（ 循环（ Kreb’s cycle)Kreb’s cycle) 柠檬酸循环（柠檬酸循环（ Citric cycle)Citric cycle)

3.1.1 3.1.1 葡萄糖的有氧分解包括三个部分：葡萄糖的有氧分解包括三个部分： GlucoseGlucose

PyruvatePyruvate

Acetal-CoAAcetal-CoA

TCA CycleTCA Cycle

NADNAD++

NADH +HNADH +H++

CoA-SHCoA-SH

COCO22

COCO22 + H + H22OO

ⅠⅠ

ⅢⅢ

ⅡⅡ

GlycolysisGlycolysis

Yielding of aceYielding of acetyl-CoAtyl-CoA

acetyl-CoAacetyl-CoA is oxidizedis oxidized

Krebs 提出三羧酸循环的观察与推理1. 肌肉悬液可以氧化二羧酸 :succcitate, fumarat

e, maltate, oxaloacetate, -ketoglutarate 和三羧酸： citrate, isocitrate, aconitrate;

2. 以上二羧酸和三羧酸促进糖和 pyruvate 的氧化 ;3. 丙二酸（ malonate ）抑制 pyruvate 的氧化；4. Oxaloacetate + pyruvate citrate5. 在丙二酸存在下，加入 fumarate, maltate, ox

aloacetate 可引起 succcitate 积累；

3.1.2 3.1.2 三羧酸循环的研究简史三羧酸循环的研究简史

3.1.2 3.1.2 三羧酸循环的研究简史三羧酸循环的研究简史

6. 6. 在丙二酸存在下，加入 oxaloacetate 可克服抑制，并且每消耗 1 分子 pyruvate, 就消耗 1 分子 oxaloacetate ；

7. 循环中每一酶促反应的速度与 pyruvate 以及 O2 的利用速度是一致的。

3.2 Pyruvate is oxidized to 3.2 Pyruvate is oxidized to acetyl-CoAacetyl-CoA

Pyruvate is transported into mitochoPyruvate is transported into mitochondriandria then oxidized to acetyl-CoA (re then oxidized to acetyl-CoA (releasing COleasing CO22) before entering the citri) before entering the citri

c acid cyclec acid cycleNADNAD++ NADH +HNADH +H++

PyruvatePyruvate Acetal-CoA + COAcetal-CoA + CO22

pyruvate dehydrogenase complexpyruvate dehydrogenase complex

3.2.1 pyruvate dehydrogenase co3.2.1 pyruvate dehydrogenase complexmplex

three enzymes

six coenzymes

complex

pyruvate dehydrogenase (E1)

dihydrolipoyl transacetylase (E2)

dihydrolipoyl dehydrogenase (E3)

CoA-SH

NAD+/NADH•H+

Lipoate ( 硫辛酸）FAD

TPP

Mg2+

Regular proteins Protein kinasePhosphate protein phosphatase

丙酮酸丙酮酸 dHEdHE 复合物的组成复合物的组成

20

5-6

60

3.2.2 The process of oxidation from 3.2.2 The process of oxidation from PPyruvate to acetyl-CoAyruvate to acetyl-CoA

活性乙醛

E2

:

Ө

E2

E2

Ө+

dihydrolipoyl

transacetylase

E3

E3

E3

E3

3.2.3 Regulation of pyruvate 3.2.3 Regulation of pyruvate dehydrogenase complex dehydrogenase complex

受蛋白激酶和磷酸蛋白磷酸酶控制――磷酸化失活 受激素调节

受能荷效应物调节激活剂

ADP, AMP E1

CoASH, NAD+ E2, E3

抑制剂 ATP, GTP E1

Acetal-CoANADH•H+ E2, E3

3.3 Tricarboxylic Acid Cycle3.3 Tricarboxylic Acid Cycle

3.3.1 Reaction process of tricarboxylic acid 3.3.1 Reaction process of tricarboxylic acid

cyclecycle

NAD+ and FAD takes the electrons NAD+ and FAD takes the electrons

during acetyl-CoA oxidationduring acetyl-CoA oxidation Each acetyl-CoA is cEach acetyl-CoA is c

ompletely oxidized tompletely oxidized to two COo two CO2, generatin, generatin

g three g three NADHNADH, one , one FADHFADH2, and one , and one GTGT

PP;; The metabolites conThe metabolites con

tain six, five or four ctain six, five or four carbons in the cycle.arbons in the cycle.

3.3.1.1 The cycle begins with the condensation of acetyle-CoA and oxaloacetate to form citrate

Catalyzed by citrate synthase (a dimer)

草酰乙酸

催化 TCA 的第一步反应，反应先生成柠檬酰 CoA ，再水解为柠檬酸，是放能反应，不可逆。是 TCA 的第一个调节酶，活性受 ATP 、 NADH 、 Succinyl CoA 及长链脂酰 CoA 的抑制，对于 TCA 是一个 rate-limitting step 。

氟乙酰 CoA 在酶的作用下可与草酰乙酸生成氟柠檬酸，顺乌头酸酶只识别柠檬酸，对氟柠檬酸没有作用，致使 TCA 中断，这种合成为致死合成 (lethal synthesis) 。在代谢研究的应用上，曾被广泛用于杀虫剂或灭鼠药的生产。 ( 现已被明令禁止 )

柠檬酸合酶 (Citric acid synthase)

合酶 (synthase) ：–催化加合反应，促使合成

合成酶 (synthetase) ：–催化连接反应，一般需要 NTP

3.3.1.2 Citrate isomerizes to isocitrate3.3.1.2 Citrate isomerizes to isocitrate via cis-aconitate via cis-aconitate

G0’ = 8.4 kJ/mol G0’ = -2.1 kJ/mol90% 4% 6%

Floride citrate is the special inhbitor of aconitase

* ** * * *

CH2-COOH

C

CH2-COOH

HO COOH(a)

(b)

柠檬酸投影式

pro-Rpro-S

R: rectusS: sinister

前手性、潜手性（ pro-chiral)

Aconitase catalysing a stereo reactionAconitase catalysing a stereo reaction

*

*

*

*

*

*

*

*

Aconitase catalysing a stereo reactionAconitase catalysing a stereo reaction

C

H COOH

C

CH2 COOH

COOH

OH

H

C

H COOH

C

CH2 COOH

COOH

H

HO

COOH

C

C

CH2

COOH

H OH

H COOH

COOH

C

C

CH2

COOH

H H

COOHHO

isocitrate citrate

isocitrate citrate

Specificity of stereochmistry of actonitase:Specificity of stereochmistry of actonitase:

Actonitase can distinguish between citrite’s Actonitase can distinguish between citrite’s

pro-R and pro-S carboxymethyl groups;pro-R and pro-S carboxymethyl groups;

Actonitase catalyzes the stereospecific trans Actonitase catalyzes the stereospecific trans

addition of OHaddition of OH-- and H and H++ across the double bo across the double bo

nd.nd.

3.3.1.3 Isocitrate undergoes oxidative de3.3.1.3 Isocitrate undergoes oxidative decarboxylation to form carboxylation to form -ketoglutarate-ketoglutarate

Reaction 4 and 5

Go’ = -20.9 kJ/mol

* * **

CO2

O

for isocitrate dehydrogenase

Positive effectors: ADP, AMP, isocitrate

Negative effectors: ATP, NADH

**

* * * *

3.3.1.43.3.1.4-ketoglutarate undergoes oxidative -ketoglutarate undergoes oxidative

decarboyxlation to form succinyl-CoA decarboyxlation to form succinyl-CoA

Catalyzed by -ketoglutarate dehydrogenase complex;

almost the same as pyruvate dehydrogenase complex;

E3 is identical, E1 and E2 are very similar.

* * * *

3.3.1.5 Cleavage of the thioester bond in s3.3.1.5 Cleavage of the thioester bond in succinyl-CoA is coupled to GTP or ATP foruccinyl-CoA is coupled to GTP or ATP for

mationmation**

（也称琥珀酸硫激酶）

3.3.1.6 Succinate is oxidized to fumarate3.3.1.6 Succinate is oxidized to fumarate

1. The enzyme catalyzes stereospecific dehydrogenation2. Malonate is the inhibitor of the reaction

琥珀酸 延胡索酸

丙二酸 琥珀酸

3.3.1.7 Fumarate is hydrated to L-m3.3.1.7 Fumarate is hydrated to L-malatealate

延胡索酸苹果酸

3.3.1.8 Oxaloacetate is regenerated 3.3.1.8 Oxaloacetate is regenerated by the oxidation of L-malateby the oxidation of L-malate

*

*

*

*

or

The citric acid cycle was confirmThe citric acid cycle was confirmed to be universal in cellsed to be universal in cells

TCATCA 循环被证明在细胞中是广泛存在的循环被证明在细胞中是广泛存在的 Details worked out by studying highly purifi

ed enzymes of the cycle in vitro;

使用高度提纯的酶在体外实验证明了这一代谢途径

Also by isotope tracer experiments in vivo;

通过体内同位素示踪法也证明了这一代谢途径

3.3.23.3.2 Survey of TCA cycleSurvey of TCA cycle 1. 总反应式 :

CH3-C-SCoA + 3NAD+ + FAD + GDP + Pi + 2H2O

2CO2 + 3NADH•H+ + FADH2 + GTP + CoA-SH

2. There are two carbon atoms in the form of acetyl-SCoA enters every cycle; and there are two decarboxylations take place in each cycle;

乙酰辅酶 A 中的二碳乙酰基进入 TCA 循环；在循环中发生两次脱羧反应

O

3. There are four dehydrogenation reactions take place in each cycle;

在循环中发生四次脱氢反应

4. There is a substrate level phosphoralation;

循环中发生一次底物水平磷酸化

3.3.23.3.2 Survey of TCA cycleSurvey of TCA cycle

5. The reactions catalyzed by aconitase and 5. The reactions catalyzed by aconitase and

fumarase is asymmetry reactions;fumarase is asymmetry reactions;

顺乌头酸酶和延胡索酸酶等催化非对称反应 6. All of the intermediary metabolisms are n6. All of the intermediary metabolisms are n

ot synthesis and decomposition netly.ot synthesis and decomposition netly.

所有的中间代谢产物在代谢中没有净合成或所有的中间代谢产物在代谢中没有净合成或净降解净降解

3.3.2 Survey of TCA cycle3.3.2 Survey of TCA cycle

3.43.4 TheThe energetics of the areable de energetics of the areable decomposotion of glucosecomposotion of glucose 葡萄糖有氧分解途径的能量计算葡萄糖有氧分解途径的能量计算

1. Acetyl in acetyl-SCoA was oxydized:

化学氧化释放的自由能： 874.9 kJ/mol

生物氧化释放的自由能： 520.9 kJ/mol

用于 ATP 合成的自由能： 354 kJ/mol

%5.409.874

354

TheThe energetics of the aerobic decom energetics of the aerobic decomposotion of glucoseposotion of glucose

2. Hydrations in TCA-cycle:2. Hydrations in TCA-cycle:

The third H2O entered in TCA cycle:

GDP + Pi = GTP + H2O

succinyl-CoA + H2O

Succidate + CoA-SH

H2O

H2O

TheThe energetics of the aerobic decom energetics of the aerobic decomposotion of glucoseposotion of glucose

3. Dehydrogenations in oxydation of glucose:3. Dehydrogenations in oxydation of glucose:

In EMP:

C6H12O6 2×pyruvate + 4H

In formation of acetyl-CoA:

2×pyruvate2 × CoA-SH

In TCA cycle: 2×acetyl-CoA + 2× 3 H2O 16H + 4CO2

2 × CoA-SH

So: C6H12O6 + 6H2O 24H + 6 CO2

6O2 12H2O

C6H12O6 + 6O2 6H2O + 6 CO2

2×acetyl-CoA + 4H +2CO2

4. Caculation of energy4. Caculation of energy

TheThe energetics of the aerobic decom energetics of the aerobic decomposotion of glucoseposotion of glucose

In EMP:

ATP 4 - 2Numbers of ATP

2NADH•H+ 2 × 3

In formation of acetyl-CoA:

2NADH•H+ 2 × 3

In TCA cycle:

3NADH•H+ × 2 3 × 3 × 2

FADH2 × 2 1 × 2 × 2

GTP × 2 1 × 1 × 2

Summery: 38

(→FADH2) (2 × 2 )

(36)

4. Caculation of energy4. Caculation of energy

The energetics of the aerobic decomThe energetics of the aerobic decomposotion of glucoseposotion of glucose

In EMP:

ATP 4 - 2Numbers of ATP

2NADH•H+ 2 × 2.5

In formation of acetyl-CoA:

2NADH•H+ 2 × 2.5

In TCA cycle:

3NADH•H+ × 2 3 × 2.5 × 2

FADH2 × 2 1 × 1.5 × 2

GTP × 2 1 × 1 × 2

Summery: 31

(→FADH2) (2 ×1.5 )

(30)

3.5 3.5 TCATCA 环的生理意义环的生理意义 1. All organic chemicals can be completely

oxidized to CO2 and H2O via a cycled pathway;

所有的有机化合物都可以通过 TCA 环彻底降解为 CO2 和 H2O

2. Plenty energy can be provided accompany the cycled pathway;

伴随着这一循环途径，大量的自由能被转换

3.5 3.5 TCATCA 环的生理意义环的生理意义 3. The intermediates in the cycle are imp

ortant sources for biosynthetic precursors;

TCA 环中的中间代谢产物是重要的生物合成的前体

TC

AT

CA

循环在合成代谢

循环在合成代谢

中的作用

中的作用

3.5 3.5 TCATCA 环的生理意义环的生理意义 3. The intermediates in the cycle are impor

tant sources for biosynthetic precursors;

TCA 环中的中间代谢产物是重要的生物合成的前体

4. The cycle is an amphibolic pathway, and is a metabolic hinge of all biochemicals

TCA 环是一个分解合成无定向途径，是所有生物化合物的代谢枢纽。

厌氧细菌（厌氧细菌（ anaerobic bacteriaanaerobic bacteria ）中的）中的 TCATCA 环环是中断的是中断的

They lack the -keto—glutarate dehydrogenase;

厌氧菌缺乏 - 同戊二酸脱氢酶

Precursors are provided for the synthesis of amino acids, nucleotides, heme etc.

合成代谢的前体通过正向或逆向反应合成

3.6 3.6 Reactions that replenish citric acid Reactions that replenish citric acid cycle intermediates cycle intermediates – anaplerotic reactions – anaplerotic reactions TCATCA 环的回补环的回补

回补反应回补反应(1)  动物肝脏和肾脏的线粒体中，丙酮酸羧化酶催化

OCCOOH CH3COCOOH + CO2 + ATP + H2O CH2COOH + ADP + Pi

Mg2+,biotin

  ΔG = 2.1 kJ/mol

(2)    原核、真核中广泛存在的苹果酸酶催化

CH3COCOOH+CO2 + NADPH+H+ Malate + NADP+

ΔG = - 3.6 kJ/mol

回回 ((添添 )) 补反应补反应 (3)    心脏、骨骼肌中， PEP 羧激酶催化 PEP + CO2 + GDP oxaloacetate + GTP

(4) 植物、细菌等， PEP 羧化酶催化 CH2CCOOH + H2O + CO2 O=CCOOH + Pi

|   O~P CH2COOH

ΔG = - 2.9 kJ/mol

(5) Asp 和 Glu 转氨形成草酰乙酸和 - 酮戊二酸；Ile,Val,Thr 及 Met形成琥珀酰 CoA ，实现回补。

丙酮酸羧化支路丙酮酸羧化支路

PEP Pyruvate acetyl-CoA

oxaloacetate

CO2 + ATP

ADP + Pi

CO2

GDP

GTP

Citrate

malate

NADH+H+

NAD+

3.7 3.7 乙醛酸循环乙醛酸循环 (Glyoxylate Cycle)(Glyoxylate Cycle)

微生物和植物可以在产乙酸或产生 acetyl CoA 的化合物中生长，因为它们存在两种酶：异柠檬酸裂解酶（ isocitrate lyase ）和苹果酸合酶（ malate

synthase ），这样可使 TCA 循环中的异柠檬酸不经脱羧而被裂解酶裂解为琥珀酸和乙醛酸，乙醛酸与另一分子 acetyl-CoA 在苹果酸合成酶作用下缩合形成苹果酸。

意义：连接糖与脂的相互转变； 协同 TCA ，造成 C4 的赢余 ;

特殊的回补方式

乙醛酸

乙醛酸(G

lyoxylate)

(Glyo

xylate)

循循

环环苹果酸合酶

苹果酸合酶

乙醛酸循环和

乙醛酸循环和T

CA

TC

A

循环的关系

循环的关系

异柠檬酸脱氢酶活性的

异柠檬酸脱氢酶活性的

调节决定异柠檬酸向乙

调节决定异柠檬酸向乙

醛酸循环还是

醛酸循环还是T

CA

TC

A

循环循环

TC

AT

CA

循环的调节

循环的调节

糖分解代谢调节

糖分解代谢调节