参考案例生化上-of enzymespart p

Binding

modes

of

enzymaticcatalysisInduced

fitLysozyme

binds

an

ionicintermediate

tightlyProperties

of

serine

proteasesAn

ionic

reaction

——has

ionicintermediates.Nucleophilic

reactionElectrophilic

reaction6.1 The

terminology

ofmechanistic

chemistryNucleophilic

reaction:A

nucleophilic

attack

or

a

nucleophilicsubstitutionThe

reactions

is

completed

by

anucleophile——has

a

negative

charge

oran

unshared

electron

pair.A

type

of

nucleophilic

substitution

(P163,6.1)Another

type

of

nucleophilic

substitution(P163,

6.2)

to

form

transition

sitate

which

isan

unstable,

high-energy

state.

It

hasastructure

between

that

of

the

reactant

andthat

of

the

product.Cleavage

reactions:(1)

Both

electros

can

stay

with

one

atom(most

reactions)

to

form

an

ionicintermediate

and

a

leaving

group;Carbanion

(P163,

6.3)Carbocation

(P164,

6.4)(2)

One

electron

can

remain

with

eachatom

(less

common)

to

formtwo

freeradicals

which

is

a

molecule

or

atom

withan

unpaired

electron

(P164,

6.5).Oxidation-reduction

reactions:They

are

central

to

the

supply

ofbiological

energy.An

oxidizing

agent——gains

electronsIf

itis

an anic

compound,thenumber

of

its

C-H

bond

is

increased.A

reducing

agent——donates

electronsDehydrogenation——the

most

common

form

ofbiological

oxidation;Addition

of

oxygen

(P164,

6.6)Removal

of

electrons6.2 Catalysts

stabilize

thansition

statesThe

rate

of

a

chemical

reactiondepends

on

how

often

reactingmolecules

collide

in

such

a

way

that

atransition

state

is

formed.The

colliding

substances

must

be

in

thecorrect

orientation

and

must

possesssufficient

energy.The

transition

state

is

an

unstablearrangement

of

atoms

and

haveextremely

short

lifetimes

(about

10-14to

10-13

s).The

activation

energy——the

energy

required

toreach

the

transition

state

from

the

ground

state.The

transition

state

occurs

at

the

peakof

theactivation

barrier.IntermediatesThe

lower

the

barrier,the

more

stable

thetransition

state

,

the

higher

theconcentratin

of

the

transition

state,

andthe

more

often

the

reaction

proceeds.Intermediates——unlike

transition

states,can

be

sufficiently

stable

to

be

detected

orisolated.

But

some

intermediates

that

aretoo

short-lived

to

be

isolated

or

detectedclosely

resemble.The

rate-determining

step(ra

imitingstep)

——

the

step

with

the

highest

energytransition

state.Catalysts

participate

directly

in

reactionsby

stabilizing

the

transition

states.Enzymes

are

catalysts

that

lower

theoverall

activation

energy

of

a

reaction

byproviding

a

multistep

pathway

(with

one

orseveral

intermediates)

in

which

the

stepshave

lower

activation

energies

thanthecorresponding

stages

in

the

nonenzymaticreaction.The

active

sites

of

enzymes

bind

not

onlysubstrates

and

products

but

also

transitionstates.In

fact,

transition

states

bind

to

activesites

mu ore

tightly

than

substrates

do.The

extra

binding

interactions

stabilize

thetransition

state,further

lowering

theactivation

energy

(P166,

Fig

6.3).6.3 Chemical

modes

of

enzymatic

catalysisTwo

major

chemical

modes

ofcatalysis

——acid-base

catalysis

andcovalent

catalysis.

They

are

sensitiveto

pH.Polar

amino

acid

residues

in

activesites.Polar

amino

acid

residues

(orsometimes

coenzymes)

that

undergochemical

changes

during

enzymeaticcatalysis

make

up

the

catalytic

centerof

the

enzyme

(P167,

table

6.1).The

following

ionizable

residues

are

foundin

active

sites

of

enzymes.Protons

transfer,

binding

SProtons

transfer,

binding

SAcyl

groups

transferAcceptor

or

donor

of

protons.Acyl

group

transferH

bonding

to

ligandsThe

pKa

of

the

ionizable

groups

of

AAs

inproteins

may

differ

from

the

values

of

thesame

groups

in

free

AAs

(P167,

table6.2).These

differences

are

usually

small

but

canbe

significant.One

can

test

whether

particular

AAsparticipate

in

a

reaction

by

examining

theeffect

of

pH

on

the

reaction

rate.

If

thechange

in

rate

correlates

with

the

pKa

of

acertain

ionic

AA,

a

residue

of

that

AA

maytake

part

in

catalysis.Acid-basecatalysisIt

is

the

most

common

catalysis

inenzymatic

reactions,

which

is

achieved

bycatalytic

transfer

of

a

proton.General

acid-base

catalysis——They

arecompleted

by

AA e

chains

(such

as

Hisat

neutral

pH)

as

donor

or

acceptor

ofprotons.A

general

base

catalysis

(a

proton

acceptor)can

assist

ractions

in

two

ways:It

can

cleave

O-H,

N-H,or

even

some

C-H

bonds

by

removing

a

proton

(P167,6.7);Participate

in

the

cleavage

of

other

bondsinvolving

carbon,

shch

as

a

C-Nbondbyremoval

of

a

proton

from

a

molecule

ofwater

(P167,

6.8).A

general

acid

(a

proton

donor)

csoassist

in

bond

cleavage

by

donating

aproton

to

an

atom.

A

covalent

bondmay

break

more

easily

if

one

of

itsatoms

is

protonated

(P168,

6.9).Covalent

catalysisAbout

20%

of

enzymes

employ

covalentcatalysis.In

covalent

catalysis,

a

substrate

or

theirpart

is

bound

covalently

to

the

enzyme

toform

a

reactive

intermediate.The

side

chains

of

AAs

can

be

either

anucleophile

(more

common)

or

anelectrophile.The

group

X

can

be

transferred

frommolecule

A-X

to

molecule

B

in

the

followingtwo

steps

via

the

covalent

ES

complex

X-E:A-X

+E

X-E

+

AX-E

+

BB-X

+

EAn

example

(P168)pHaffec zymatic

ratesEnzymatic

reactions

are

effected

by

pH.Sensitivity

to

pH

usually

reflectsanalteration

inthe

ionization

state

of

AAs

inits

active

site.The

pH

optimum

of

an

enzymeThe

pH-rate

profile

of

anenzyme——

mostisbell-sh d

curve.An

example,papain(P169,Fig

6.4、Fig

6.5)Relative2

8

10

pHA:

pepsin;

B:

Glucose-6-phosphataseactivityABThe

pHoptimum一些酶的最适pH值酶最适

pH胃蛋白酶1.8过氧化氢酶7.6胰蛋白酶7.7延胡索酸酶7.8核糖核酸酶7.8精氨酸酶9.8最适pH：在一定条件

下,酶具有最大的催化活性的pH值。不同酶的最适温度也不一样。动物酶的最适温度一般在35-40℃，植物酶为40-50℃。少数酶可达60℃以上，如：细菌淀粉水解酶的最适温度90℃以上。嗜热细菌：Taq聚合酶最适温度70℃，93℃不失活。5.

The

temperatureoptimum

of

an

enzyme6.4

Diffusion-controlledreactionsDiffusion-controlled

raeactions

——theformation

of

the

EScan

be

theslowest

step

(the

rate-determiningstep)

(P171,

table

6.3).

The

overallrate

of

the

reaction

may

approach

theupper

limit

forcatalysis.Only

a

few

types

of

chemical

reactionscan

proceed

this

quickly,

includingassociation,

some

proton

transfers,and

electron

transfers

reactions.Two

examples

ofdiffusion-controlledraeactions.

Triose

phosphateisomerase

(TPI).Aproton

transferreactionIt

has

two

ionizableactive-siteresidues

——

GLu165and

His95(P172,

Fig

6.6).Triose

phosphateisomeraseThe

kinetice

research

on

TPI.All

four

step

barriers

are

approxima y

same(P173,

Fig

6.7).The

Kcat/Km

is

4×108

M-1

s-1,

close

to

the

rateof

aduffusion-controlled

reaction.

Itappearsthat

this

enzyme

has

evolved

to

achieve

itsum

possible

efficiency.The

mutant

of

TPI

——Glu165

to

Asp165

hassimilar

Km,

but

greatlydecreasing

kcat

towild-type

TPI

so

that

it

catalyzes

the reaction

about1000

times

slowerthan

wild-type.This

experiment

showed

that

Glu165

is

essential.The

mutagenesis

changed

a

duffusion-controlledenzyme

to

a

more

typical

enzyme

——rapid

bindingof

substrates

and

slower

catalysissteps2.

Superoxide

dismutase

(SOD)It

catalyzes

the

very

rapid

removal

of

the

toxicsuperoxide

radical

anion

(P174,

6.19)It

is

an

even

faster

catalyst

than

TPI.Itskcat/Km

is

near

2×109

M-1

s-1,

faster

thantypical

diffusion

rates.Because

its

negatively

charged

substrate

isatracted

by

a

posivively

charged

electric

fieldnear

the active

site

——

Cu2+

and

4

hydrophilicAAs

(two

Glu,

one

Lys

and

one

Arg).

The

electricfield

around

the

SOD

active

site

enhances

therate

of

formation

of

ES

about

30-fold.6.5 Binding

modes

of

enzymatic

catalysisThe

proper

binding

ofactivesite

and

S

providesnot

only

substrate

and

specificitybut

also

mostof

the

catalytic

power

of

enzymes.There

are

two

catalytic

modes

based

on

bindingphenomena.(1)

The

proximity

effect.

Formultisubstratereactions,

the

collecting

and

correct

positioningof

S

inactivesite

raises

their

effectiveconcentrations.(2)

Transition-state

stabilization.The

bindingof

transition-state

to

enzymesshould

be

moretighter

than

that

of

substrates

or

products

toenzymes.1.

The

proximity

effectIt

can

increase

reaction

rates

more

than

10000folds.It

make

the

reaction

of

twomolecules

tivesite

as

an

intramolecular

or

unimolecularreaction.

It

enhanced

the effective

molarity.Effective

molarity

=

k1(s-1)/k2(M-1

s-1)K1

is

the

rate

constant

of

single

molecularreaction.

K2

is

the rate

constantof

bimolecularreaction.

The

effective

molarity

are

not

realconcentrations.

It

indicate

how

favorablyreactive

groups

are

oriented.The

proximity

effect

is

illustrated

bysomeexperiments

which

compared

anonenzymaticbimolecular

reaction

to

a

series

of

icallysimilar

intramolecular

reactions.2.

Weak

binding

of

substrates

to

enzymesThe

binding

of

S

to

enzymes

can

not

beextremelytight,

that

is

,

Km

can

not

be

extremely

low.

Why

?If

a

substrate

were

bound

extremely

tightly,

itcould

take

just

asmuch

energy

to

reach

ES*

fromES

as

is

required

to

reach

S*

from

S

in

thenonenzymatic

reaction

(P177,

Fig6.9).Km

appear

to

be

optimized

by

evolution

foreffective

catalysis

——

low

enough

that

proximityis

achieved,

but

high

enoughthat

the

ES

is

nottoo

stable.In

fact,

most

Kmare

on

the

order

of

10-4M,

anumber

that

indicates

weak

binding

of

the

S.

Inan

ES,

not

all

partsof

the

substrate

are

bound.3.

Transition-statestabilizationTransition-state

stabilization

explains

nearlyallthe rate

acceleration

of

enzymes

and

is

nowconsidered

the

major

factor.Some

transition

states

may

bindto

their

enzymesmore