Professor Wu Yaosheng

2009-10

BiochemistryDept. of Biochemistry and Molecular Biology

2

還沒有來得及準備好接受這一地的金黃， 秋天就這樣悄無聲

息的來到了我們的身邊。

Chapter 9

Regulation of Metabolism

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Main Contents

1. Metabolic Regulation at Cell Level

2. Metabolic Regulation at Hormone Level

3. Regulation of Metabolism at Integral Level

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Key Points and difficulties

◆ Some important metabolism molecules

◆ Mutual relationship of carbohydrate, TG, Pr

◆ Key enzymes and their distribution

◆ Regulation levels and fashion of substance metabolism

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1.Mutual interknit among various metabolism pathways

Sugar

Lipid

Protein

H2O

Salt

Vitamin

各种物质代谢之间互有联系，相互依存。

Digestion Absorption

Middle metabolism

Waste excretion

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Introduction Characteristics of Substance Metabolism

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2. Metabolism processes regulated constant finely

Subtle regulation mechanisms to regulate metabolism intensity, direction, velocity

Inside and outside of circumstances

To influence organism metabolism

To fit in with the change of circumstances

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3. Various tissues and organs have themselves metabolism characters

Different structures

Different enzymes and contents

Different organs

Different metabolism pathways

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4. Each common metabolism pool

For example：

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Sugar digested and absorbed

glycogen degradation

gluconeogensis

Blo

od

sug

ar Va

riou

s tis

su

es

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5. ATP is the common form for energy store and

utilization

Nutriment decomposition

To release energy

ADP+Pi

ATP

Directly supply energy

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6. NADPH can supply the reduction equation for anabolism

For example ：

Acetyl CoA

NADPH + H+

Fatty acids, cholesterol

Pentose phosphate pathway

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Questions

1. How to relate carbohydrate metabolism with lipid or protein metabolism by some important interim molecules?

2. What are the important significances of ATP during substance metabolism?

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What are metabolic interrelationships?

Section One

Metabolic Regulation at C

ell Level

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1.1 Distribution of Enzymes in Cells

•代谢途径有关酶类常常组成多酶体系，分布于细胞的某一区域 。

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Metabolic pathways Distribution Metabolic pathways Distribution

Glycolysis Cytosol Oxidation phosphorylation

Mitochondrion

Citric acid cycle Mitochondrion Protein synthesis ER

Pentose phosphate pathway

Cytosol Urea synthesis Mitochondrion, cytosol

Gluconeogenesis Cytosol DNA synthesis Nucleus

Glycogenesis and glycogenolysis

Cytosol mRNA synthesis Nucleus

Fatty acid β-oxidation Mitochondrion tRNA synthesis Nucleoplasm

Fatty acid synthesis Cytosol rRNA synthesis Nucleus

Respiratory chain Mitochondrion Heme synthesis Cytosol, Mitochon.

Phospholipid synthesis Endoplasmic reticulum

Hydrolytic enzymes Lysosome

Cholesterol synthesis ER, Cytosol Bilirubin synthesis ER, cytosol

Distribution of enzymes in main metabolic pathways

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Distribution of enzymes in main metabolic pathways

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Significances

◆To avoid interference among enzymes in different metabolic pathways

◆ To be benefit to harmonious operation of enzymes

Compartmentalization of enzymes in cells

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1.2 Multienzyme system, Multifunctional Enzymes, and Isoenzymes

1.2.1 Multienzyme System and Multifunctional Enzymes

Multienzyme system is an enzyme complex assembled by several different functional enzymes. For example, pyruvate dehydrogenase complex

Multifunctional enzyme is an enzyme with different enzymatic functions in a single polypeptide. For example, fatty acid synthase system

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The fatty acid synthase complex has 7 active sites:

Acetyl CoA-ACP transacetylase (AT)-ketoacyl-ACP synthase (KS)Malonyl CoA-ACP transferase (MT) -ketoacyl-ACP reductase (KR)-hydroxyacyl-ACP dehydratase (HD)Enoyl-ACP reductase (ER)Acyl carrier protein (ACP)

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1.2.2 Isoenzymes

Enzymes catalyzing the same reaction with different components and different physicochemical properties are named as isoenzymes. For example, LDH

H H

H H

H H

H M

H H

MM

H

MM

M

MM

MM

LDH1

(H4)LDH2

(H3M)

LDH3

(H2M2)LDH4

(HM3)

LDH5

(M4)

lactate dehydrogenase, LDH isoenyzmes19

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Example Two

BB BB BBMM MM MM

CK1(BB) CK2(MB) CK3(MM)

brain cardiac muscle skeleton muscle

肌酸激酶 (creatine kinase, CK) 同工酶

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1.3 Basic Manners of Metabolic Regulation at Cell Level

1.3.1 Rate-Limiting Enzyme and Rated-Limiting Step

Definition for rate-limiting enzyme:

An enzyme with relatively low activity catalyzing the relatively low reaction speed for control the rate of the whole pathway is named rate-limiting enzyme.

A B C D E F GE1 E2 E3 E4 E5 E6

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Rate-limiting enzymes of some metabolism pathways

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Metabolism pathway Rate-limiting enzymes

Glycolysis HK , PFK-1, PK

P.P.P G6PD

Gluconeogenesis Pyr carboxylase, PEP carboxykinse, FBPase, G6Pase

Cictric acid cycle Citrate synthase, Isocitrate DHase, α-KG DHase

Glycogenesis Glycogen synthase

Glycogenolysis Glycogen phosphorylase

Triacylglycerol hydrolysis Triacylglycerol lipase

FA synthesis Acetyl CoA carboxylase

Ketogenesis HMG CoA synthase

Cholesterol synthesis HMG CoA reductase

Urea synthesis Argininosuccinate synthase

Heme synthesis ALA synthase

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1.3.2 Feedback Regulation

The end-products in metabolism pathways often affect the activities of the initial enzymes.

Feedback regulation is one of the finest acting manners of regulatory enzymes.

Positive feedback: F-1,6-BP to 6-FPK-1

Glycogen phosphorylase

Glucogenolysis : Gn G1P G6P G

(—)

(+)Glycogen synthase

UDPG

Negative feedback: most key enzymes

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1.3.3 Substrate Cycle

Substrate cycle is the reversible interconversion between two substrates catalyzed by distinct enzymes for unilateral reactions.

F-1,6-2P

F-6-P

ADP ATP

Pi

FPK-1

Fructose biposphatase-1

AMP F-2,6-2P(+)

(–)

(+)

(–)

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In a chain reaction, when an enzyme is

activated, other enzymes are activated in

turn to bring primal signal amplifying.

1.3.4 Cascade Reactions

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Adenyly cyclase （ inactive ）

hormones （ glucagon 、 epinephrine ） + receptor

cAMP

PKA(inactive)

Phosphorylase b kinase

PKA(active)

Phosphorylase b Phosphorylase a-P

Phosphorylase b kinase-P

Adenyly cyclase（ active ）

ATP

inactive active26

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腺苷环化酶 （无活性）

腺苷环化酶（有活性）

激素（胰高血糖素、肾上腺素等） + 受体

ATP cAMP

PKA( 无活性 )

磷酸化酶 b 激酶

糖原合酶 糖原合酶 -P

PKA( 有活性 )

磷酸化酶 b 磷酸化酶 a-P

磷酸化酶 b 激酶 -P

Pi

磷蛋白磷酸酶 -1

Pi Pi 磷蛋白磷酸酶 -1 磷蛋白磷酸酶 -1

–

–

–磷蛋白磷酸酶抑制剂 -P

磷蛋白磷酸酶抑制剂 PKA （有活性） 27

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1.4 Regulation of Enzymatic Activity in Cells

1.4.1 Allosteric Regulation ( rapid regulation ）

when some metabolites combine reversibly to an regulating site of an enzyme and change the conformation of the enzyme, resulting in the change of enzyme activity.

◆◆ allosteric effectors allosteric effectors

◆◆allosteric enzymeallosteric enzyme

◆◆ allosteric siteallosteric siteAllosteric activator

Allosteric inhibitor 28

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Some allosteric enzymes and their effectors in metabolism pathways

Metabolism Allosteric enzymes Activator Inhibitor

GlycolysisHK G-6-P

6-FPK-1 AMP, ADP, F-1,6-BP, F-2,6-BP

Citrate, ATP

Pyruvate kinase F-1,6-BP ATP, alanine

Citric acid cycleCitrate synthase ADP ATP, citrate, NADH

Isocitrate dehydrogenase ADP ATP, Ca2+

Gluconeogenesis Pyruvate carboxylase Acetyl CoA ADP

F-1,6-bisphosphatase Citrate AMP, F-2,6-BP

Glycogenolysis Glycogen phophorylase b

AMP, G-1-P, Pi ATP, G-6-P

Glycogenesis Glycogen sythase G-6-P

FA biosynthesis Acetyl CoA carboxylase Citrate, isocitrate Long-chain fatty acyl-CoA

Cholesterol biosynthesis

HMG-CoA carboxylase Cholesterol

AA metabolism L-glutamate dehydrogenase

ADP, leucine, methionine

ATP, GTP, NADH

Purine synthesis PRPP amidotransferase PRPP AMP, ADP, GMP, GDP,

Pyrimidine synthesis Aspartate transcarbomoylase

CTP

Heme synthesis ALA synthase Heme

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Key points: An allosteric enzyme is regulated by its effectors (activator or inhibitor). Allosteric effectors bind noncovalently to the enzyme. Allosteric enzymes are often multi-subunit proteins. A plot of V0 against [S] for an allosteric enzyme gives a si

gmoidal-shaped curve. The binding of allosteric enzyme with an effector will induce a conformational change Does not consume energy

General Properties of Allosteric Enzymes

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T state R state(high activity) (low activity)

FDP

FDP

FDP

FDPFDP

FDP

FDP

FDP

AMP

AMP

AMP

AMP

(allosteric inhibitor)AMP

Glyceraldehydes-3-phosphateFA –carrier protein

(allosteric activator)

Allosteric effect of fructose-1,6-biphosphatase

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1.4.2 Covalent Modification (rapid regulation ）

It means the reversible covalent attachment

of a chemical group.

Types of Covalent Modification:Types of Covalent Modification: phosphorylation / dephosphorylation adenylylation/deadenylylation methylation/demethylation acetylation/deacetylation －－ SH / SH / －－ SS －－ S S , etc

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Protein-OH

Protein-O-P=O

O-

O-

ATP

ADP

Protein kinase

H2O

Pi

Protein phosphatase

The reversible phosphorylation and dephosphorylation of an enzyme

Covalent Modification

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Regulation of covalent modification in enzyme activities

PFK-1 Phosphorylation/dephosphorylation Inactivity/activity

Pyr DHase Phosphorylation/dephosphorylation Inactivity/activity

Pyr decarboxylase Phosphorylation/dephosphorylation Inactivity/activity

Glycogen phosphorylase Phosphorylation/dephosphorylation Activity/inactivity

Phosphorylase b kinase Phosphorylation/dephosphorylation Activity/inactivity

Protein phosphatase Phosphorylation/dephosphorylation Inactivity/activity

Glycogen synthase Phosphorylation/dephosphorylation Inactivity/activity

Triacylglycerol lipase Phosphorylation/dephosphorylation Activity/inactivity

HMG CoA reductase Phosphorylation/dephosphorylation Inactivity/activity

Acetyl CoA carboxylase Phosphorylation/dephosphorylation Inactivity/activity

Enzyme Reactive type Effect

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The activity state of an enzyme modulated can

interconvert reversely

Change of a covalent bond catalyzed by E, and

can be modulated by hormones

The modification is a rapid, reversible and eff

ective and amplified by cascade reaction

The most common is the phosphorylation or

dephosphorylation. Enzymes----protein kinases

or phosphatases

Key points:

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PP

PP

P

P

2ATP 2ADP

2Pi

Phosphorylase b kinase

phosphatase

Phosphorylase b(dimer)

Inactivity

Phosphorylase a(dimer)

High activity

Phosphorylase a(tetramer)

Activity

Covalent modification of phosphorylase

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1.5 Regulation of Enzyme Level in Cells(Genetic Control)

The amount of enzyme present is a balance between the rates of its synthesis and degradation.

The level of induction or repression of the gene encoding the enzyme, and the rate of degradation of its mRNA, will alter the rate of synthesis of the enzyme protein.

Once the enzyme protein has been synthesized, the rate of its breakdown (half-life ) can also be altered as a means of regulating enzyme activity.

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1.5.1 Induction and repression of E Pr Synthesis

Induction: the activation of enzyme synthesis.Repression: the shutdown of enzyme synthesis.

Genetic control of enzyme leverl means to controlling the transcription of mRNA needed for an enzyme’s synthesis.

In prokaryotic cells, it also involves regulatory proteins that induce or repress enzyme’s synthesis.

Regulatory proteins bind to DNA, and then block or enhance the function of RNA polymerase. So, regulatory proteins may function as repressors or activators.

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Repressor

Repressors are regulatory proteins that block transcription of mRNA, by binding to the operator that lies downstream of promoter.

This binding will prevent RNA polymerase from passing the operator and transcribing the coding sequence for the enzyme.------Negative control.

Regulatory proteins are allosteric proteins. Some special molecules can bind to regulatory proteins and alter their conformation, and then affect their ability to bind to DNA.

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Structural geneOperator gene Promotor

repressor gene

I

NH2

For example: lac operon

Z Y

repressor protein

mRNA

A

mRNA

When no lactose:

RNA polymeras

e

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lactose

Structural gene

repressor gene

I

NH2

Z Y

repressor protein

mRNA

A

mRNA

When lactose presents:

P O

RNA polymeras

e

NH2

NH2

ZYA

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Inducers Inducers promote the transcription of mRNA.

OP

Structural gene

RNA polymeras

e

activator-binding site

Activator is an allosteric protein which is unable to bind to promoter to transcribe relative genes directly in eukaryotes.

Activator

When no inducer:

mRNA

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Structural gene

P O

RNA polymeras

e

activator-binding site

activator

When inducer:

mRNA

inducer

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Bacteria also Use Translational Control of Enzyme Synthesis

The bacteria produces antisense RNA that

is complementary to the mRNA coding for the

enzyme.

When the antisense RNA binds to the mR

NA by complementary base paring, the mRNA

cannot be translated into protein.

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1.5.2 Degradation of Enzyme Proteins

Cellular enzyme proteins are in a dynamic state with change of enzyme synthesis and degradation so that ultimately determine enzyme level at any point in time.

In many instances, transcriptional regulation determines the concentrations of specific enzyme, with enzyme proteins degradation playing a minor role.

In other instances, protein synthesis is constitutive, and the amounts of key enzymes and regulatory proteins are controlled via selective protein degradation.

In addition, it also involves the abnormal enzyme proteins ( biosynthetic errors or post-synthetic damage).

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There are two pathways to degrade enzyme protein in cells:

1. Lysosomal pathway

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ATP independent

2. Proteasome pathway

ATP, Ubiquitin dependent

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Questions

1. Which one of the following metabolism pathways is not present in cytoplasm?

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A. Glycolysis B. Phosphate pentose pathway C ． Glycogenesis and glycogenolysis D ． Fatty acid β-oxidation E ． Fatty acid synthesis

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Questions

2. All gluconeogenesis, ketone body biosynthesis and urea synthesis exist in

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A. Heart B ． Kidney C ． Brain D ． Liver E ． Muscle

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Can you fill in these blanks?

Substrate cycle is the reversible interconversion between two substrates catalyzed by distinct enzymes for unilateral reactions.

F-1,6-2P

F-6-P

ADP ATP

Pi

FPK-1

Fructose biposphatase-1

AMP F-2,6-2P(+)

(–)

(+)

(–)

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ATP

(+)

Fructose biposphatase-1

(–) (–)

(+)

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Questions

1. Why some persons who are easely drunk can turn to endure alcohol after they have experience to drink wine?

2. Why some persons who need hypnotics ( 安眠药） would become more and more dependent to drugs?

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Section Two

Metabolic Regulation at Ho

rmone Level

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Hormones are generally secreted by endocrine glands, travelled by blood stream to specific target cells.

By these mechanisms, hormones regulate the metabolic processes in various organs and tissues; facilitate and control growth, differentiation, reproductive activities, learning and memory; and help organisms coping with changing conditions and stresses to around environment.

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Hormonal regulation depends upon the transduction of the hormonal signal across the plasma membrane to specific intracellular sites, particularly the nucleus.

Many steps in these signal across the signalling pathway involve phosphorylation of Ser, Thr, and Tyr residues on target proteins.

According to receptor’s location in a cell, hormones are divided into two classes:

Hormones act on cell membrane receptors

Hormones act on intracellular receptors

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5454

Hormones act on cell membrane receptors

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Hormones act on intracellular receptors

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2.1 Regulation of Hormones to Receptors on Cell Membrane

Hormones act on membrane receptors, as the first messenger, to activate various signal transduction pathways that mobilize various second messengers-----cAMP, cGMP, Ca2+, IP3 , DG that activate or inhibit enzymes or cascade o

f enzymes in specific ways.

The first messengers: Peptide or protein hormones: GH, Insulin, etc Amino acid derivatives: epinephrine, norepinephrine

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RＲ

H

AC

γαβ

GDPαGTP

βγ

Adenylate cyclase

AC

ATP

cAMP

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Hormone receptor

Ｇ protein Enzyme

The second messenger

Protein kinase

Enzyme or other protein

Biological effects

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2.2 Regulation of Hormones to Receptors in Cells

Hormones to act on intracellular receptors:

Steroid hormones: Glucocorticoids

Mineralocorticoids

Vit D

Sex hormones

Amino acid derivatives: T3, T4

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Hormone receptor

Ｇ protein Enzyme

The second messenger

Protein kinase

Enzyme or other protein

Biological effects

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Can you give some examples?

Section Three

Regulation of Metabolism

at Integral Level

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Living in a constantly changing environ

ment, human must have the ability to adapt

ing to the environment.

The metabolism of body has to be regul

ated through neurohumoral pathways to sa

tisfy energy needs and to maintain homeos

tasis of the internal environment.

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Why and how?

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3.1 Metabolism Regulation in Starvation

3.1.1 Starvation in Short-term (1-3 days)

Glycogen reserve

Blood Glucose

Insulin

glucagoncorticosteroid

a series of metabolic changes

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(1) Protein Metabolism

Protein

Amino acid gluconeogenesis

deamination

Pyruvatetransamination Alanine

Blood

degradation

Alanine

Pyruvate

Glucose

transamination

Protein degradation ↑, Amino acid Glucose

Muscle

Liver

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(2) Carbohydrate Metabolism

Gluconeogenesis

Liver : 80%

Renocortical : 20%

Lactic acid 30%

Glycerol 10%

Amino acids 40%

Tissue utilize glucose

In brain , glucose is still the main fuel

source.66

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(3) Triacylglycerol Metabolism

Fat mobilization

Fatty acid Ketone bodies

Heart Skeletal muscle

Part

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3.1.2 Change of Metabolism in Long-term Starvation ( >7 days)

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Amino acid , but Glu deamination

Starvation in Long-term(1) Protein Metabolism

Muscle protein degradation

Urea

NH3 Acidism( 酸中毒）In urine

( by ketosis 酮症 )

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(2) Carbohydrate Metabolism

( almost equal to that in liver )

In kidney : Gluconeogenesis

Lactic acidPyruvate

The main materials of gluconeogenesis in li

ver:

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(3) Triacylglycerol Metabolism

Fat mobilization

Fatty acidKetone bodies

Skeletal muscle: FA as an energy source to ensure that adequate amounts of ketone bodies are available in brain.

Brain: gradually adapts to using ketone bodies as fuel.

This may reduce utilization of glucose and gluconeogenesis of amino acid, so decrease the breakdown of protein.

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After starvation in Long-term, if the person is given a big meal with a lot of meat and wine in short time, what case would occur?

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3.2 Metabolism Regulation in Stress

injury

pain

frostbite

oxygen deficiency

toxicosis

infection

out-of-control rage

Excitation of sympathetic nerves

Adrenal medullary/cortical hormones

Epinephrine, glucagons, growth hormone

Insulin

Metabolism of carbohydrates

lipids change

proteoins

Effect:Stimulus

Catabolism Anabolism

Stress is a tense state of an organism in response to unusual stimulus.

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(1) Change of Carbohydrate Metabolism

Hyperglycemia catecholamine

glucagon growth hormone corticosteroid

Insulin

GluconeogenesisGlycogenolysis

Blood glucoseIf exceeds renal thre

shold of glucose (8.96 mmol/L)

GlucosuriaStress hyperglycemia Str

ess glucosuria74

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(2) Change of Triacylglycerol Metabolism

AdrenalineNoradrenalineGlucagon

Fatty acidKetone bodies

Fat mobilization

Tissue utilize FA as energy

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(3) Change of Protein Metabolism

Protein hydrolysis

Amino acid: as material for Gluconeogenesis

Urea synthesis

Equilibrium of negative nitrogen

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Stress

Sympathetic excitation

Adrenal cortex/ medulla hormone

TG hydrolysisLipocyte

Liver

Gluconeogenesis

glucose

Glycerophosphate

Glycogenolysis

Ketogenesis Pyruvate Ureogenesis

FA LA Alanine NH3

Urea

Blood vessel

Kidney

Glucosuria

FA LA Glucose

Glycerophosphate

Alanine

Muscle

Muscle glycogenolysis

Protein degradation

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Questions

1. Which one of substance change in blood is incorrect under stress ?

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A. Glucose increase B. Free fatty acid increase C ． Amino acid increase D ． Ketone body increase E ． VLDL increase

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Questions

2. When hungry, the false statement about substance metabolism alternation is

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A ． Gluconeogenesis enhancement B. Triglyceride mobilization enhancement C ． Ketone body synthesis enhancement

D ． Insulin secretion increase E. Glucagon secretion increase

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Questions

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1.How does Ala turn to be glucose in vivo? When does this case occur？

2. How does carbohydrate metabolism and amino acid metabolism be modulated in liver cells to adapt with those in skeleton muscles and in cardiac muscle?

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Questions

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3. How to compare allosteric regulation with chemical modification?

4. Use several examples to explain some diseases involved with abnormal metabolism.

5. What changes of metabolism in body would occur in long-term starvation?