Chapter 5 Enzymes

Jia-Qing Zhang Ph.DBiochemistry department Medical school Jinan UniversityMar. 2007

What are enzymes?

Enzymes are proteins which act as catalysts

Catalyst :

A catalyst is something which by its very

nature increases the rate of a reaction and

remain uncharged at the end of reaction.

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catalyst

enzyme

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Enzymes control and regulate the

various metabolic activities inside living

cells.

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In the absence of an enzyme:

whereas in its presence the rate can be

increased up to 107 fold.

The definition of enzymes?

Enzymes are powerful and highly effectual

biocatalyst produced by living tissues which

increase the rate of reactions that occur in the

tissue.

What are enzymes made up of ?

Almost all enzymes are make up of proteins

In 1982s, Thomas Cech discovered RNA possessing catalytic activity, were called ribozymes.

In 1995, Jack W. Szostak lab reported a DNA fragment with ligase function, termed Deoxyribozyme

So biocatalysts have enzyme, ribozymes, deoxyribozyme

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Section 5.1 Structure and function of Enzymes

5.1.1 Structure of Enzymes

5.1.1.1 Composition of Enzymes Molecules Enzymes are claasified into

Simple enzyme (e.g. urease , protease , lipase etc.)

Conjugated enzyme. Most enzymes are conjugated.

Simple enzymes are all proteins,

Conjugated enzymes include protein component and

Non protein constituents,

Holoenzyme = apoenzyme + cofactorAn anpoenzyme is the protein part

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Cofactors commonly seen are 1. Metal ions ; iron, magnesium, cobalt manganese Metal ions tightly link to enzyme are known as

metalloenzyme

those link between E and S(substrate) are called metal activated enzyme(Table 5-2).

2. Low M.W. organic compounds. Vitamins often play their roles in H+ ， electron

and other chemicals transfer when they take

place in metabolism as coenzyme （ Tab5-1 ）

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Cofactor

is divided into:

Coenzyme Prosthetic group

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Differences between coenzyme and prosthetic group

1.Coenzyme linked to apoenzyme loosly and can be aparted from holoenzyme by dialysing,while prosthetic group linked tightly and can not be separated that way.

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2.Coenzyme leave apoenzyme after catalyzing a reaction,

Prosthetic group can not depart from holoenzyme.

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5.1.1.2 Monomeric enzymes,

Oligomeric enzymes and Mult

ienzyme complex

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monomeric sarcosineoxidase

Monomeric enzymes

Monomeric enzymes only contains tertiary structure

trypsin

22Oligomeric enzyme

Oligomeric enzymes

contains two or more polypeptide chains associated b

y noncovalent forces.

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Multienzyme complex is that different enzymes

catalyze sequential reactions in the same path

way are bound together. PDC

Multienzyme complex

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5.1.2 Active site of an enzyme

Active center: the region of an enzyme that contains some chemical groups for binding substrate and for catalyzing the conversion of substrates to products.

All groups in active site are termed the essential groups.

Essential group: some chemical groups connected to activity of enzyme.They are classified into binding group and catalytic group.

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底 物 活性中心以外的必需基团

结合基团

催化基团

活性中心 目 录

Active sites are

usually located in

clefts between

domains or

subunits or

indentations on

surface of proteins

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5.1.3 Structure and function of enzymes

5.1.3.1 The primary structure of enzymes and Its function

Simliar catalyzed function means structure homology.

eg. Chymotrypsin trypsin and elastase

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Chymotypsin and trypsin

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5.1.3.2 The spatial structure of enzymes and its function

The catalyzed activity of enzymes also depends on the conformation of enzymes.

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Section 5-2 Nomenclature and Classification of Enzyme

In Naming an enzyme substrates are stated first

ending –ase is affixed

Enzymes are grouped into six classes according to the nature of the reactions

4) Peroxidases

5) reductase

Catalyze cleavege of bonds by addition of water

Catalyxe racemization of optical or geometric

Isomers and intramolecular oxidation-reduction

reactions

Ligases are usually referred to as synthetases

Carboxylase

5.3 Properities and catalytic mechanisms of enzymes

5.3.1 properities of enzyme catalytic reactions

5.3.2 catalytic mechanisms of enzymes

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Properities of enzyme catalytic reactions

1).High catalytic activity of enzymes:

(a) Enzymes can decrease activation energy i.e.

molecules are activated using activation energy.

By decreasing activation energy,enzymes promotes

chemical reactions

(fig 5-5) .

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(b) After Product has formed, free Enzymes are released and reused.

(c) Enzyme can make a reaction 103-107 times faster than uncatalyzed,

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2) Highly specificity of enzymes:

Specificity refers to the ability of an enzyme to discriminate between two competing substrates.

The specificity can be divided into 3 types:

(a)Absolute specificity: the extreme selectivity of E that

allows it to catalyze only a single S.

e.g chymotrypsin hydrolysis peptide bond having aroma

tic AA, trypsin catalyse those having basic AA.

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(b) Group specificity(relative specificity):

Enzymes atc on a group of related substrates

e.g. Hexokinase catalyzes the phosphorylation of

glucose, mannose, fructose, glucosamine

Esterase catalyse the break down of ester

bonds .

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(c) Optical(stereo) specificity:

e.g. LDH hydrolyzes L-lactic acid, but not

D- lactic acid

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3). Under control :enzyme can be regulated

Regulated in different levels: biosynthesis, allo

steric regulation covalent modification isoenzy

mes.

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5.3.2 catalytic mechanisms of enzymes

Enzymes increase the rate of reaction by lowering the

activation energy?

Then, how to decrease the energy?

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Formation of ES complex and Induced-fit hypothesis

The enzyme reversibly combines with its substrate

to form an ES complex, that subsequently break down

to product, regenerating the free enzyme ；

E + S ----- ES ------- P + E

P

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Lock and Key Theory:

The specific action of an enzyme with a single substrate

can be explained using a Lock and Key analogy first po

stulated in 1894 by Emil Fischer. In this analogy, the lock

is the enzyme and the key is the substrate. Only the corr

ectly sized key (substrate) fits into the key hole (active

site) of the lock (enzyme).

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The following experimental evidence show the lock

and key theory is rigid model and can not explain the

ES complex well. For this reason a modification

called the induced-fit theory has been proposed

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Induced Fit Theory:

The induced-fit theory assumes that the substrate

plays a role in determining the final shape of the

enzyme and that the enzyme is partially flexible. I

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summaryenzyme\enzyme_binding.swf

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5.3.2.3 several factors contribute to enzyme catalysis

2. Electrostatic effects

3. Acid-base catalysis

4. Covalent Catalysis

1. Proximity effects and orientation arrange:

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1. Proximity effects and orientation arrange:

Chemical reactions are based on molecular

collision,proximity and orientation lead to correct

direction for collision.

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2. Electrostatic effects The strength of electrostatic interactions can reduce

the attractive forces and increase the chemical

reactivity of the substrate.

3. Acid-base catalysis

Enzymes is usually ampholyte, can provide acid or basi

c enviorment, partial proton tranfer from an acid or to a

base lowers the activation energy. the rate of reaction c

an often be promoted by adding or removing a proton.

4. Covalent Catalysis

Accelerates reaction rates through the transient formation

of a catalyst-substrate covalent bond

Section 5.4 Kinetics of Enzyme-catalyzed reactions

KineticsThe branch of chemistry that is concerned with the

rates of change in the concentration of reactants in a

chemical reaction.

Enzyme kinetics

Study of the rate of change of reactants to products and factors by which rate of reaction is influenced, such as substrates, activators, inhibitors, pH and temperature.

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Enzyme activity can be expressed by kinetic properties:

Internation units(IU) one IU is defined as the amount of enzyme that produces 1umole of product per minute.

1 katal(kat)1 katal(kat) is equal to the amount of enzyme that converts one mole of substrate to product per second.

5.4.1

The effect of substrate on the rate of e

nzyme-catalyzed reactions

5.4.1.1

The basic conditions for discussing the

effect of substrate on the rate of enzyme-

catalyzed reactions

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5.4.1.2 Rectangular hyperbola plot of initial

velocity V0 versus substrate concentration

[S]

[S][S]

VVVmaxVmax

At low [s], V0 increases linearly with increase in [S]

[S][S]

VVVmaxVmax

At higher [S], V0 increases nonlinearly with increase in [S]

[S][S]

VVVmaxVmax

At very higher [S], V0 increases is vanishingly small with increase in [S]

5.4.1.3 Formulation of the Michaelis-menten equation

Michaelis-menten equation, a mathematical equ

ation expressing the hyperbolic relationship between

the initial velocity, Vo, and the substrate concentratio

n, [S].

The enzymes-catalyzed reaction process may be

summarized as follow:

k2

k3

k4

,so k4 ingored

k2

k3

k3

k3

k2

[1]

k3 [1]

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k3

k3

k2

[1]

At this point, it’s important to draw your attention to two assumption mentioned above:

one is [s] >>[E].

the other is , it’s assumed that the system is in steady

state, that the ES complex is being formed and broken

down at the same rate. So that

overall [ES] is constant.

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Formation equal to breakdown, then

K1[E][S] = (k2 + k3) [ES]

The formation of ES will depend on the k1 and the

availability of E and S.

So

Rate of ES formation = k1 [E][S]

The breakdown of [ES] can occur in two ways, either the conversion of S to P, or non-reactive dissociation of S from ES complex.

Rate of ES breakdown = (k2 + k3) [ES]

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[ES]

k1

K2 + k3=

[E][S]

By rearranging

K1[E][S] = (k2 + k3) [ES]

[2]

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[ES]=

[E][S]

As the name implies, these 3 rate terms k are constants, so M

ichaelis actually combine them into one term, this new consta

nt is termed the michaelis constant and is written Km

k1

K2 + k3=Km

Substituting the Km into equation [2]

[2]

Km

[ES]

k1

K2 + k3=

[E][S]

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The total amount of enzyme in the system must be the same throughout the experiment, but it can either be free (unbound) E or in complex with substrate, ES.  If we term the total enzyme Et, this relationship can be written out:

t

[3]

Substituting this definition of [E] back into equation [3] gives us:

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t

t

[E] = [Et] –[ES]

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First of all, open the bracket so that the [E] and [ES] are separately multiplied by [S]

t

Next, multiply each side by KM, this gives us:

t

t

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Then collect the two [ES] terms together on the same side. This gives:

t

Then because both terms on the right-hand side are multiplied by [ES] we can collect them together into a bracket:

Dividing both sides by (KM + [S]) now gives us:

t

t

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t

Substituting this left-hand side into Equation 1 in place of [ES] results in:

3

t0 [4]

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The maximum rate, which we can call Vmax, would be achieved when all of the enzyme molecules have substrate bound. Under conditions when [S] is much greater than [E], it is fair to assume that all E will be in the form ES. Therefore

[Et] = [ES]

Notice that k3[Et] was present in equation 4, so we can replace this with Vmax, giving a final equation:

Thinking again about Equation 1, we could substitute the term Vmax for v and [Et] for [ES]. This would give us:

Vmax = k3[Et]

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0

This final equation is actually called the Michaelis-Menten equation.

3t

0 [4]

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5.4.1.4 The Significance of Km and Vmax

Let us consider the case when V is exactly half of Vmax. Under those circumstances, the Michaelis-Menten equation could be written:

On dividing both sides by Vmax this becomes:

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Multiplying both sides by (KM + [S]) gives:

And then multiplying both sides by 2 further resolves the equation to:

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[S] on the right-hand side is the same as [S] + [S], so we

can take away one [S] from each side.  Thus when the

rate of the reaction is half of the maximum rate:

If we now reconsider the graph that present at

the start of this class it could be written:

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Significance of Km:

1. Km is an important constant depends on E , S structure and reaction environment.

2. Km is equal to the [S] at which the reaction occurs at half of the Vmax rate. 

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3. Sometime(When k2 is much greater than k3), km c

an indicates the affinity of the ES complex.

Some enzymes have more than 2 substrates, higher K

m, weaker binding of S and E.

e.g. Glucose and Fructose are substrates of hexokina

se , Glu has less Km , higher affinity to hexokinase, in c

ontrast, Fru. has higher Km and less affinity.

If E has more than 2 substrates, the least Km is th

e optimum

Significance of Vmax:

Vmax, the maximal initial velocity, is obtained only whe

n all the enzymes is in the form of the ES complex, from

which it follows that:

From above equation, Vmax = k3 [Et]

3

2

3

2

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K3 is the turnover number of enzymes.

Turnover number of an enzyme

is the number of substrate molecules converted into

product by an enzyme molecule in a unit of time when

the enzyme is fully saturated with substrate.

, Vmax

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Double reciprocal plot ( Lineweaver-Burk plot)

In order to obtain accurate Km, the Michaelis formul

a can be turned into a linear formula by up-side-dow

n the numerator / denominator so as to obtain 1/ Vm

ax at y axis and 1/Km at x axis.

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up-side-down the numerator / denominator, give us

1/v0 : y

1/[s]: x

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The temperature at which enzymes operate at

maximal efficiency

Optimum temperature

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At a pH above this optimum, the enzyme's activity will be reduced and therefore the reaction rate will be lowered; at a pH below this optimum, the enzyme's activity again will be reduced and lower reaction rates result

5.4.5 The effect of inhibitors on the rate of enzyme-catalyzed reactions

Inhibitor (I)

An agent that can decrease enzyme activity without

causing denaturation of enzyme.

Inhibitor can be classified into:

(1)Irreversible inhibitors:

(2) Reversible inhibitors: Competitive, un-competitive, non-competitive

Irreversible inhibition

Irreversible inhibitors bond and destroy a functional

group in an enzyme that is essential for the enzyme

activity

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•In most cases forms a covalent link with the enzyme

•Permanently renders the protein inactive

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AChE

Inhibitor--Organic phosphorus

Inhibition of AChE by organic phosphorus

PAM

Organic phosphorus (public Hazards in a

gricultural drugs) : usually binds to active sit

e of acetylcholine esterase.

ACh

-noncompetitive

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Chemicals that resemble an enzyme's normal substrate an

d compete with it for the active site. Block active site fro

m the substrate.

If reversible, the effect of these inhibitors can be overco

me by increased substrate concentration.

Competitive inhibition

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In competitive inhibition, both inhibitor and substrate can

bind to enzyme and form two independent complexes.

Only ES degrades to products: EI is considered a 'dead-

end'. Because the inhibitor binds to the active site, the

substrate cannot (and vice versa), so there cannot be

an ternary ESI complex.

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Mechanism and Features:

1. I and S compete the active site;

2. Which one occupies the active site depends

on the affinity of S / I affinity to E and concentr

ations between I and S.

3. Km increase ( affinity decrease), Vmax unc

hanged

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Mathematical analysis

For those who are interested in Maths

only

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km

Noncompetitive inhibition

Noncompetitive inhibitors :Enzyme inhibitors that do

not enter the enzyme's active site, but bind to another

part of the enzyme molecule. Causes enzyme to

change its shape so the active site cannot bind

substrate.

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The Vmax is decreased without a change in the K

m for the substrate

The inhibition cannot be overcome by increaseing

the substrate concentration.

Features of non-competitive inhibition:

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Uncompetitive inhibition

Uncompetitive inhibitors bind only to the ES

complex and not the free enzyme.

Features:

Decrease of Km and Vmax.

summary

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5.4.6 Activators of enzymes

Activator: Agents increase the activity of enzyme

or make the inactive form become active form are c

alled Activator of enzyme.

They are mostly metal ions(Mg2+,K+,Mn2+),also som

e anion(Cl-)and organic compounds.

Most anions are necessary to enzyme activity,they

are called essensial activator.Of course there are n

on-essential activator,Cl- is so to ptyalin .

Section 5.5 Regulation of Enzyme Activity

•Allosteric regulation

•Covalent modification

There are two major strategies for regulating enzymes:

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The mechanism of Allosteric regulation

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General properties of allosteric enzymes

1. The activities of allosteric enzymes are charged by metabolic activators and inhibitors, which seldom resemble the substrates or products. So allosteric regulation is not inhibition.

2. Modulators bind noncovalently to enzymes.

3. Allosteric enzymes almost possess quaternary structure.

4. Allosteric enzymes often display sigmoid plots of the the reaction V versus S

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Covalent modification

The covalent attachment of another molecule can

modify the activity of enzymes and many other

proteins.

In these instances, a donor molecule provides a

functional moiety that modifies the properties of

the enzyme.

Most modifications are reversible.

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Phosphorylation and dephosphorylation are the

most common but not the only means of covalent

modification.

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Other forms of modification:

Acetylation

Methylation

nucleoside modification.

Significance of modification :

Rapidly change between the active and inactive form

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Zymogen

Several enzymes are produced and stored as

inactive precursors called zymogens

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e.g. LDH(lactate dehydrogenase) LDH

1,2,3,4,5 are HHHH, HHHM, HHMM, HM

MM and MMMM . i.e. LDH isozymes are

tetramers formed by 2 sets of subunits.

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Significance :

In the heart, LDH1 (4 H) H=heart, catalyzes Lactate convert to pyruvate for energy supply

In muscles, LDH5 ( 4M ) M=muscle, convert pyr to Lact. For energy storage.

Clinical diagnosis using isozyme. E.g. when heart attack(infarction) happens, enzymes release from injured cells to the blood showed different enzyme(isozyme ) pattern.

Isozyme pattern: different isozymes appear as a peak sooner or later followed by the progress of the disease.

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Summary

1.Definition:

active site, allosteric regulation, Covalent modification,

Zymogen, Isoenzymes, Coenzyme, prothetic group,

2. Explain the kinetic significance of Km and Vmax

3. What would affect the activity of an enzyme, and how?

4. What are differences betweeen competitive ,

noncometitive and uncompetitive inhibition?

6. How does the value of Km and Vmax change when adding competitive, noncompetitive and uncompetitive inhibitor respectively?