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The Organic Chemistry of

Enzyme-Catalyzed Reactions

Revised Edition

Professor Richard B. SilvermanDepartment of Chemistry

Department of Biochemistry, MolecularBiology, and Cell Biology

Northestern !niversity

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"he #rganic Chemistry ofEn$yme%Cataly$ed Reactions 

Chapter &

 En$ymes as Catalysts

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'or pu(lished data regarding any en$yme see)http)**.(renda%en$ymes.info*

NomenclatureEnzyme Names

  EC Number

  Common/ Recommended Name  Systematic Name  Synonyms  CAS Registry Number

Reaction & Specificity  Pathway  Catalysed Reaction

  Reaction Type  Natural Substrates and Products  Substrates and Products  Substrates  Natural Substrate  Products  Natural Product  Inhibitors  Coactors  !etals/ Ions  Acti"ating Compounds  #igands

Functional Parameters  $m %alue  $i %alue  pI %alue  Turno"er Number  Speciic Acti"ity  p& 'ptimum  p& Range  Temperature 'ptimum  Temperature Range

Isolation & Preparation  Puri(cation  Cloned  Renatured  Crystallization

Organism- related information  'rganism

  Source Tissue  #ocalization

Stability  p& Stability

  Temperature Stability  )eneral Stability  'rganic Sol"ent Stability  '*idation Stability  Storage Stability

Enzyme Structure  Se+uence/ SwissProt lin,  -.Structure/ P.0 lin,  !olecular 1eight  Subunits  Posttranslational !odiication

Disease & References  .isease  Reerences

pplication & Engineering

  Engineering  Application

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+hat are en$ymes, and ho do they or-

'irst /isolation0 of an en$yme in &122 Ethanol added to a3ueous e4tract of malt

5ielded heat%la(ile precipitate that as

utili$ed to hydroly$e starch to solu(le sugar6precipitate no non as amylase

&171 % 89hne coined term enzyme % means/in yeast0

&1:1 % Duclau4 proposed all en$ymes shouldhave suffi4 /ase0

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En$ymes % natural proteins that cataly$e

chemical reactions 'irst en$yme recogni$ed as protein as ;ac(ean urease

Crystalli$ed in &:

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En$ymes have molecular eights of severalthousand to several million, yet cataly$e

transformations on molecules as small ascar(on dio4ide and nitrogen

'unction (y loering transition%state energiesand energetic intermediates and (y raisingthe ground%state energy

Many different hypotheses proposed for hoen$ymes cataly$e reactions

Common lin of hypotheses) en$yme%cataly$ed reaction alays initiated (y theformation of an enzyme-substrate ?or E •SAcomplex  in a small cavity called the active site

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&1: % Lock-and-key hypothesis % 'ischer

proposed en$yme is the loc into hich thesu(strate ?the eyA fits Does not rationali$e certain o(served

phenomena)

Compounds having less (uly su(stituents oftenfail to (e su(stratesSome compounds ith more (uly su(stituents

(ind more tightly

Some en$ymes that cataly$e reactions (eteento su(strates do not (ind one su(strate untilthe other one is (ound

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&:@1 % Induced-fit hypothesis proposed (y8oshland)

+hen a su(strate (egins to (ind to an en$yme,interactions induce a conformational change

in the en$ymeResults in a change of the en$yme from a lo

catalytic form to a high catalytic form

nduced%fit hypothesis re3uires a fle4i(le activesite

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Concept of fle4i(le active site stated earlier (yPauling ?&:=A)

ypothesi$ed that an en$yme is a fle4i(letemplate that is most complementary tosu(strates at the transition state rather thanat the ground state

"herefore, the su(strate does not (ind mosteffectively in the E•S comple4

 s reaction proceeds, en$yme conforms (etter

to the transition%state structureTransition-state stabilization results in rate

enhancement

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#nly a do$en or so amino acid residues maymae up the active site

#nly to or three may (e involved directly insu(strate (inding and*or catalysis

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+hy is it necessary for en$ymes to (e so large-

Most effective (inding of su(strate resultsfrom close pacing of atoms ithin protein

Remainder of en$yme outside active site isre3uired to maintain integrity of the active site

May serve to channel the su(strate into theactive site

 ctive site aligns the or(itals of su(strates andcatalytic groups on the en$yme optimally forconversion to the transition%state structure%%called orbital steering 

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En$yme catalysis characteri$ed (y tofeatures) specificity  and rate acceleration

 ctive site contains amino acid residues andcofactors that are responsi(le for the a(ovefeatures

Cofactor , also called a coenzyme, is anorganic molecule or metal ion that is essentialfor the catalytic action

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Specificity of En$yme%Cataly$ed Reactions "o types of specificity) ?&A Specificity of (inding

and ?

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K s =

E + S E   . E   . E + P

k 2

k -1

k 1

k -1

k 1

S P

Scheme &.&

k on

k off 

Michaeliscomple4

+hen k < FF k %&,

k 

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aEigen, M.6 ammes, G.G. !d" Enzymol" 1963, #$ , &.

Table 1.1. Examles of T!rno"er #!mbersa

Enzyme T!rno"er n!mber  

k cat $s-1%

papain &>car(o4ypeptidase &><

acetylcholinesterase&>

2

inases &>2

dehydrogenases &>2

aminotransferases &>2

car(onic anhydrase &>=

supero4ide dismutase &>=

catalase &>7

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K m is the concentration of su(strate thatproduces half the ma4imum rate

K m is a dissociation constant , so the smallerthe K m the stronger the interaction (eteen Eand S

k cat*K m is the specificity constant  % used toran an en$yme according to ho good it isith different su(strates

!pper limit for is rate of diffusion ?&>: M%&s%&AK m

k cat

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  o does an en$yme release product soefficiently given that the en$yme (inds the

transition state structure a(out &>&<

 times moretightly than it (inds the su(strate or products-

 fter (ond (reaing ?or maingA at transitionstate, interactions that match in the transition%state sta(ili$ing comple4 are no longer present.

"herefore products are poorly (ound, resulting in

e4pulsion. s (onds are (roen*made, changes in electronic

distri(ution can occur, generating a repulsiveinteraction, leading to e4pulsion of products

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E S comple4'igure &.&#on-co"alent interactions

electrostatic

?ionicAC

O

O

+

RNH 3

ion-dipole R

C NH3

R'

Oδ

δ  +

διπολε−διπολε   Ρ 

 Χ Ο

Ρ∋

Οδ

δδ δ

Η 

Η−βονδ ινγ  

Ο

ΡΧ Ο Η  

Ο

Η 

χηαργετρανσφερ

 

Α

∆

∆

Α

ηψδροπηοβιχ

 

ΟΡΧ

Ο

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HGI J %R"lnK e3

f K e3 J >.>&, HGI of %@.@ cal*mol neededto shift K e3 to &>>

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Specific 'orces nvolved inES Comple4 'ormation

'igure &.<

NH3

O

OH

CH

3

COCH

2

CH

2

NMe

3

+

+

δ

−

δ

+

δ

−

διπολε−διπολεδ

+

ιον−διπολε

Ο

Ο

ιονιχ

E4amples of ionic, ion%dipole, and dipole%dipoleinteractions. "he avy line represents theen$yme active site

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%(onds

  type of dipole%dipoleinteraction (eteen K%

and 5) ?N, #A

'igure &.2

%(onds

ydrogen (onding in the secondary structure ofproteins) α%heli4 and β%sheet. 

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Charge "ransfer Comple4es

+hen a molecule ?or groupA that is a goodelectron donor comes into contact ith amolecule ?or groupA that is a good electronacceptor, donor may transfer some of itscharge to the acceptor 

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ydropho(ic nteractions

+hen to nonpolar groups, each surrounded(y ater molecules, approach each other, theater molecules (ecome disordered in anattempt to associate ith the ater moleculesof the approaching group

ncreases entropy, resulting in decrease inthe free energy ?∆%° J ∆& °%T ∆S°A

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van der +aals 'orces

 toms have a temporary nonsymmetricaldistri(ution of electron density resulting ingeneration of a temporary dipole

"emporary dipoles of one molecule induceopposite dipoles in the approaching molecule

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Binding Specificity

Can (e a(solute or can (e very (road Specificity of racemates may involve ES comple4

formation ith only one enantiomer or EScomple4 formation ith (oth enantiomers, (utonly one is converted to product

En$ymes accomplish this (ecause they are chiralmolecules ?mammalian en$ymes consist of only

L%amino acidsA

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Binding specificity of enantiomers

Scheme &.<

EnzL  + ( R,S ) EnzL + EnzL R S 

diastereomers

Resolution of a racemic mi4ture

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Binding energy for ES comple4 formation

ith one enantiomer may (e much higherthan that ith the other enantiomer 

Both ES comple4es may form, (ut only oneES comple4 may lead to product formation

Enantiomer that does not turn over is said toundergo nonproductie binding 

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Steric hindrance to (inding of enantiomers

'igure &.

OOCNH

3

H

OOC NH3

H

A B

S R

Leu

Basis for enantioselectivity in en$ymes

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Reaction Specificity

!nlie reactions in solution, en$ymes can shospecificity for chemically identical protons

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'igure &.@

R R'

R R'

Ha

Hb

B

-

enzyme

En$yme specificity for chemically identicalprotons. R and R′ on the en$yme are

groups that interact specifically ith R andR′, respectively, on the su(strate.

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Rate cceleration

 n en$yme has numerous opportunities toinvoe catalysis)

  Sta(ili$ation of the transition state

  Desta(ili$ation of the ES comple4  Desta(ili$ation of intermediates

Because of these opportunities, multiple

steps may (e involved

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'igure &.= &>&>%&>& fold typically

E+S

E+P

ESEP

Catalyzed

Uncatalyzed

Reaction Coordinate

A

Uncatalyzed

Enzyme Catalyzed

Reaction Coordinate

 

B

Effect of ?A a chemical catalyst and?BA an en$yme on activation energy

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En$yme catalysis does not alter the e3uili(riumof a reversi(le reaction6 it accelerates attainmentof the e3uili(rium

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a "aen from Rad$ica, .6 +olfenden, R. Science 199&, #'( , :>.( "aen from orton, .R.6 Moran, L..6 #chs, R.S.6 Ran, .D.6 Scrimgeour,8.G. )rinciples of *iochemistry 6 Neil Patterson) Engleood Cliffs, N, &::2.

Table 1.'. Examles of Enzymatic Rate (cceleration

Enzyme #onenzymatic rate

  )non $s-1%

Enzymatic rate

  )cat $s-1%

Rate acceleration  )cat *)non

cyclophilina %< &.2 4 &>B .= 4 &>@

car(onic anhydrasea &.2 4 &>%& &>= 7.7 4 &>=

chorismate mutasea %@ @> &.: 4 &>=

chymotrypsin( 4 &>%: 4 &>%< &>7

triosephosphateisomerase(

= 4 &>%7 < 4 &>2 2 4 &>:

fumarase( < 4 &>%1 < 4 &>2 &>&&

etosteroid isomerasea &.7 4 &>%7 =.= 4 &>B 2.: 4 &>&&

car(o4ypeptidase a 2 4 &>%: @71 &.: 4 &>&&

adenosine deaminasea &.1 4 &>&> 27> &<

urease( 2 4 &>%&> 2 4 &>B &>&B

alaline phosphatase( &>%&@ &>< &>&7

orotidine @O%phosphatedecar(o4ylasea

%&= 2: &. 4 &>&7

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Mechanisms of En$yme Catalysis 

 ppro4imation Rate enhancement (y pro4imity

En$yme serves as a template to (ind the

su(strates Reaction of en$yme%(ound su(strates

(ecomes first order 

E3uivalent to increasing the concentration ofthe reacting groups

E4emplified ith nonen$ymatic model studies

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Scheme &.2

CH3COAr

O O

CO

C

O

H3C+ CH3COO-

CH3+ ArO-

Second%order reaction of acetate itharyl acetate

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OAr

O

O

O

-

OAr

O

O

O

-

OAr

O

O

O

-

OAr

O

O

O

O

O

-

Relative rate  ( k rel

)

1  M 

-1

  s

-1

220 s

-1

5.1 x 10

4

 s

-1

2.3 x 10

6

 s

-1

1.2 x 10

7

s

-1

Decreasing rotational and

translational entropy

+ CH3

COO

-

OAr

Effective Molarity (EM)

5.1 x 10

4

 M 

2.3 x 10

6

 M 

1.2 x 10

7

 M 

220  M 

"a(le &.2. Effect of ppro4imation on Reaction Rates

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Covalent Catalysis

Scheme &.anchimeric assistance

Most common

Cys ?SASer ?#Ais ?imida$oleALys ?N

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Scheme &.@

SCl

SOH

S+

1.2

HO--Cl-

 nchimeric assistance (y a neigh(oring group

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Model Reaction for Covalent Catalysis

Scheme &.=

Early evidence to support covalent catalysis

O

O

18O

O 18OCH

3C

18O

18OH

O

OH

18O18

+Ar

H2O

H2OO-

(-ArO-)

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General cid*Base Catalysis

"his is important for any reaction in hich protontransfer occurs

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'igure &.7catalytic triad

"he catalytic triad of α%chymotrypsin. "hedistances are as follos) d & J

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Scheme &.7

HN

NH

NHR'

SerO

H

R1

O R2

O

R

N N H

His

-OOC Asp

Charge relay system for activation of an active%site serine residue in α%chymotrypsin

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pK a values of amino acid side%chain groups ithinthe active site of en$ymes can (e 3uite differentfrom those in solution

Partly result of lo polarity inside of proteins

Molecular dynamics simulations shointeriors of these proteins have dielectric

constants of a(out

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Basic group in a nonpolar environment has aloer pK a 

pK a of a (ase ill fall if ad;acent to other(ases

 ctive%site lysine in acetoacetate

decar(o4ylase has a pK a of @.: ?pK a insolution is &>.@A

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"o inds of acid*(ase catalysis)

Specific acid  or specific base catalysis -catalysis (y a hydronium ?2#QA or hydro4ide?#%A ion, and is determined only (y the p

%eneral acid+base catalysis % reaction rateincreases ith increasing (uffer concentrationat a constant p and ionic strength

Eff t f th ( ff t ti ?A

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'igure &.1

Specific acid*(ase catalysis General acid*(ase catalysis

k  k 

[Buffer] [Buffer]

pH 7.9

pH 7.3

pH 7.9

pH 7.3

A   B

Effect of the (uffer concentration on ?Aspecific acid*(ase catalysis and ?BA

general acid*(ase catalysis

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Scheme &.1

Specific cid%Base Catalysis

O

C OEt

poornucleophile

weak electrophile

++H3C

EtOHCH3COOHH2O

ydrolysis of ethyl acetate

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Scheme &.:

 laline hydrolysis of ethyl acetate

O

COHH3C

O

COC2H5H3C

O

CO-H3C

+ +

strongnucleophile

C2H5O-

HO-

C2H5OH

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Scheme &.&>

O

COHH3C

OH

COC2H5H3C

OH

COC2H5H3C

O

COC2H5H3C

+

+

++

strongelectrophile

H3O+

H2O

C2H5OH

 cid hydrolysis of ethyl acetate

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Scheme &.&&

B

+

H

R Y

O

H OHB:

Simultaneous acid and (ase en$yme catalysis

(ase catalysis

acid catalysis

En$ymes can utili$e acid and (asecatalysis simultaneously

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Simultaneous acid*(ase catalysis is the reason for

ho en$ymes are capa(le of deprotonating eacar(on acids

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Scheme &.&<

Simultaneous acid and (ase en$yme catalysisin the enoli$ation of mandelic acid

Ph

HaHOOHb

O

PhO-

O

HaHO

Ph

HaHOOHb

OHc

Ph

HaHOOHb

OHcPh

HO

O-

OHb

Ph

HO

OHc

OHb

pKE = 18.6

+

+

pKa ~ 7.4pKa = 6.6

± Hc+   ± Ha+

pKE = 15.4

pKa = 22.0

± Ha+

pKa ~ -8

± Hc+

pKa = 3.4

± Hb+

1.3  1.4

1.51.6

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Lo,-barrier hydrogen bonds % short ?F

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X R

H

O

H

B:

BH

X R

HO

:B

B+H

R

O

R = H, alkyl, SR'

O–

OX

H H

M+

BH

O–

OX H

M+

B

O

O

H

M+

BH

B

H

B

B:

B:

BH

-HX

A

B

Scheme &.&2

lo%(arrier%(ond

/ea0(ase

/strong0acid

/strong0(ase

/ea0 acid

lo%(arrier%(ond

stronger acidneeded

#ne%(asemechanismsyn%elimination

car(o4ylic acids

&,%elimination of β%su(stituted ?A aldehydes,etones, thioesters and ?BA car(o4ylic acids

"o%(asemechanismanti %elimination

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Scheme &.&

ElcB mechanism % not relevant 

X R

H

O

H

X R

O

B+H

R

O

B:

Base cataly$ed &,%elimination of β%su(stitutedcar(onyl compounds via an enolate

intermediate ?ElcB mechanismA

Needs acid or metal catalysis

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 lternative to Lo%Barrier ydrogen Bond

Scheme &.&@

R

H

O

H

R' R

O

R'

H

B: B+

H

Electrostatic en$yme catalysis in enoli$ation

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Electrostatic Catalysis

Scheme &.&=

o4yanion hole

HN

NH

HN

NH

NH

HN

NH

HN

O

O

O O

OO

O

R"

R'

RR"

R'

R

O

++

also could be aH bond or dipole

Electrostatic sta(ili$ation of the transition state

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Desolvation

  E4poses su(strate to loer dielectric

constant environment  E4poses ater%(onded charged groups for

electrostatic catalysis

  Desta(ili$es the ground state

"he removal of ater molecules at the active siteon su(strate (inding

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Scheme &.&7

Strain Energy

k 1.+

k 1., J &>1

O

P

O HO

-OP

O

O- OO--O

O

P

O

O--O

CH3CH3

OP

O-O

-O

CH3

CH3

HO

1.7   1.8

-OH -OH

 laline hydrolysis of phosphodiesters

nduced 'it ypothesis

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'igure &.:

nduced 'it ypothesisputting strain energy into the su(strate

E ti Eff t f E C t l i

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'igure &.&>

Energetic Effect of En$yme Catalysis

mportance of ground state desta(ili$ation

Mechanisms of En$yme Catalysis % porpho(ilinogen synthase

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H

Lys252

NH2

NH2

O

COO-

NH2

O

COO-

NH

NH-Lys252

COO-

NH2

COO-B:

NH

NH-Lys252

COO-

NH2

COO-

NH

COO-COO-

H

NH2

NH-Lys252

B

B:

H

:B

NH

COO- COO-

NH2

HNH

COO- COO-

NH2

+ +

..

+

+

+

..

:

ZnB(Cys)4

Lys252

NH

NH2

O

COO-

ZnB(Cys)4

H :B

Lys252

NH

NH2OH

COO-

ZnB(Cys)4

Lys252

NH

H2N

COO-

NH2

O

COO- Lys252

NH

N

COO-

HH

:B (X)3ZnA

HO

(X)3ZnA

HO

H

(X)3ZnA

HO

(X) Z

HO

(X) Zn

HO

strain energyelectrostatic catalysis

approximationcovalent catalysis

base catalysis

strain energyelectrostatic catalysis

base catalysis

base catalysis

acid catalysis

base catalysis

base catalysis

approximation

approximation

(X3)ZnA (X3)ZnA