Enzyme Cat Alys Is

7/25/2019 Enzyme Cat Alys Is

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Enzyme Catalysis


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Objective

To understand how enzymes

work at the molecular level.

Ultimately requires total structuredetermination, but can learn much through

biochemical analysis.


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To e E!"lained

Specificity For specific substrates

Amino acids residues involved Catalysis

Amino acids involved

Specific role(s) Regulation


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#actors $%%ecting Enzyme $ctivity


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"&


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E%%ects o% "& on Enzyme $ctivity

Binding of substrate to enzyme

onization state of !catalytic" aminoacid residue side c#ains

onization of substrate

$ariation in protein structure


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Tem"erature

Relative

Activity

ba

%emperature


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'nhibitors

Covalent

Reversible rreversible

(on)covalent& reversible


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$ctive *ite

inding and Catalysis


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eneral Characteristics

%#ree dimensional entity

,ccupies small part of enzyme volume

Substrates bound by multiple *ea-interactions

Clefts or crevices

Specificity depends on precise arrangementof atoms in active site


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-odels

ock and /ey -odel0t#e active site e.ists!pre/formed" in t#e enzyme prior to

interaction *it# t#e substrate+ 'nduced #it -odel& t#e enzyme undergoes

a conformational c#ange upon initialassociation *it# t#e substrate and t#isleads to formation of t#e active site+


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'denti%ication and

Characterization o% $ctive *ite

*tructure& size s#ape c#arges etc+

Com"osition& identify amino acidsinvolved in binding and catalysis+


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Catalytic *ite2e.g. Chymotry"sin3

01 C1 C 01

,

0 C

catalytic site

complementary

2CA%A85SS2

17,

,


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1robing the *tructure o% the

$ctive *ite

-odel *ubstrates


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-odel *ubstrates2Chymotry"sin3

17,(R,1)

01 C1 C0

R

01,

C

acyl transfer to 17,aromatic

side c#ain

peptide bond


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)amino grou"4

ood *ubstrate5

17C C 01

7

,

R (,C19)


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*ide Chain *ubstitutions

ood *ubstrates

Cyclohe!yl t)butyl)

C19

C19

C19


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$rginase

H2N

C

NH

(CH2)3

CH COOH3N

NH2

H2O

NH3

(CH2)3

CH COOH3N

H2N

C

O

NH2

+

+

-

+

ureaornit#inearginine

+-

+


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ood Com"etitive 'nhibitors

NH3

NH

NH3

(CH2)3

CH COOH3N

NH3

(CH2)4

CH COOH3N

O

(CH2)2

CH COOH3N

CH

NH2

-+

(

ornithine

(

+-

++

+

-

(

canavaninelysine

+


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1oor Com"etitive 'nhibitors

$ll Three Charged rou"s are 'm"ortant

019

(C17)9

C17190

019

(C17)9

17C C,,

C19

(C17)9

C1 C,,190 /

/

a/aminovaleric acid putrescine

(l


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'denti%ying $ctive *ite $mino

$cid +esidues


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Covalent 'nactivation

6iiso"ro"yl 1hos"ho%luoridate

nactivates Chymotry"sinby forming a =&=covalent adduct to Serine=>?+

odoacetic acid inactivates +ibonucleasebyreacting *it# 1is=7and 1is==>+

CH O P O CH

CH3

CH3

CH3

CH3

F

O


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$%%inity abeling(;eneral Approac#)

3ositioning ;roup

Reactive ;roup

5Binding Site

:

:


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$%%inity abeling(%osyl/8/p#enylalanine c#loromet#yl-etone)

nactivates Chymotry"sinby forming a=&= covalent adduct to 1istidine?@

O S O

NH

CHCH2

CH3

Reactive

;roup

3ositioning

;roup

O

C

O

CH2Cl


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Tra""ing o% Enzyme)ound 'ntermediate

2Chymotry"sin3

mplicates Ser=>?

in catalytic mec#anism+

CTCH2OH O2N O O C CH3

O

*er789

O2N

CTCH2O C CH3

O

O

2acyl2 enzyme

stable at p1 9

p/nitrop#enylacetate

+

O

p/nitrop#enol


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Catalytic -echanisms


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-echanisms o% Catalysis

Acid/base catalysis

Covalent catalysis

etal ion catalysis

3ro.imity and orientation effects

3referential binding (stabilization) oft#e transition state


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$cid)ase Catalysis

/eto)Enol Tautomerization

RC CH3

O

RC

OH

CH2

etone 4nol


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Uncatalyzed +eaction


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eneral $cid Catalysis


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eneral ase Catalysis


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eneral $cids

C,,1 019

C17 ,1

C17 ,1

S1

10 01

C17


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eneral ases

C,, 017

S10 0&

C17


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Figure 11-10

+ibonuclease $

N

N

O

O

OH

O

O

CH2

O

PO O

O

N

N

N

N

O

NH2

OH

CH2OP

O

O

O

PO O

O

Adenosine

ridine+ibonuclease $


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Figure 11-10 part 1

-echanism o% +(ase $


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Figure 11-10 part 2

-echanism o% +(ase $


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Covalent Catalysis(3rinciple)

*low

17, AB DDE A,1 B1

A/B 4/1 DDE 4/A B1

4/A 17, DDE A/,1 4/1

#ast


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Covalent Catalysis(C#ymotrypsin)

C% C17 ,1 ,70 ,, C C19

,

C% C17 , C C19

,

C% C17 ,1

Ser=>?

, ,,70

C% C17 , C C19

,

1, C C19

,

:rate limiting: 17,

fast

%ast

(OTE0 (ew +eaction 1athway


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-etal 'on Catalysis

-etalloenzymes& contain tig#tlybound metal ions for catalytic activity

-etal)activated enzymes& looselybound metal ions from solution

C#arge stabilization

ater ionization

C#arge s#ielding


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-etalloenzymes

Catalytically essential (tig#tly bound)&

Fe7 Fe9 Cu7 n7 and Co7

Structural metal ions& 0a and Ca7

Bot#& g7

and Gn7


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1ro!imity and Orientation E%%ects

Rate of a reaction depends on&

0umber of collisions 4nergy of molecules

,rientation of molecules

Reaction pat#*ay (transition state)


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1ro!imity

; < k=$>=>

=$> and => < ?7@- on enzyme sur%ace


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Page 336

iomolecular +eaction o% 'midazole

with ")(itro"henylacetate(ntermolecular)


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Page 336

'ntramolecular +eaction o%

'midazole with ")(itro"henylacetate(ntramolecular)

'ntramolecular +ate < AB! 'ntermolecular +ate


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Orientation

A BBA

C


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Figure 11-14

eometry o% an *(A +eaction


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*tabilize Transition *tate

1,

R

Br

5

R

R

Br

5

R

G

6

2strain2 2stabilized2

1, C

R

::

CR


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Page 338

*teric *train in Organic

+eactions

+eaction +ate0 +


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Figure 11-15

E%%ect o% 1re%erential

Transition *tate inding


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Page 339

1roline +acemase(planar transition state)


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Page 339

Transition *tate $nalogs o% 1roline

inding < 7D! versus1roline


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*erine 1roteases

Chymotry"sinTry"sin

Elastase

etc.


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/inetic $nalysis o% Chymotry"sin2&ydrolysis o% ")nitro"henylacetate3


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-echanism o% Chymotry"sin

CTCH2OH O2N O C CH3

O

CTCH2O C CH3

O

CTCH2OH

OO2N

CTCH2O C CH3

O

HO C CH3

O

:rate limiting:++ H2O

+fast

+

%ast

")nitro"henolate


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'denti%ication o% the Catalytic +esidues2+eaction o% Chymotry"sin with 6'1#3

*"ecial +eactivity o% *erine789


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'denti%ication o% the Catalytic +esidues2+eaction o% Chymotry"sin with T1C/3

$%%inity abeling

)+ay *tructure o% ovine Try"sin


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)+ay *tructure o% ovine Try"sin2+ibbon 6iagram3


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Figure 11-26

$ctive *ite +esidues o% Chymotry"sin2Catalytic Triad3


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Figure 11-29 part 1

Catalytic -echanism o% the

*erine 1roteases

Catalytic Triad


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Figure 11-29 part 2


*erine 1roteases


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Figure 11-29 part 3


*erine 1roteases


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Figure 11-29 part 4


*erine 1roteases


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Figure 11-29 part 5


*erine 1roteases


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Figure 11-29 part 5


*erine 1roteases


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Figure 11-30a

Transition *tate *tabilization in

the *erine 1roteases


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Figure 11-30b

Transition *tate *tabilization in

the *erine 1roteases


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-echanism o% Chymotry"sin

CTCH2OH O2N O C CH3

O

CTCH2O C CH3

O

CTCH2OH

OO2N

CTCH2O C CH3

O

HO C CH3

O

:rate limiting:+

+ H2O

+fast

+

%ast

")nitro"henolate

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Enzyme Cat Alys Is