The Organic Chemistry of Enzyme-Catalyzed Reactions

Chapter 2

Group Transfer Reactions: Hydrolysis, Amination,

Phosphorylation

Hydrolysis Reactions

Amide Hydrolysis

Peptidases (proteases if protein hydrolysis involved) catalyze the hydrolysis of peptide bonds

Scheme 2.1

NH3 CH C

O

NH COO- NH3

R1

CH

R2

CH C

O

NH C

R3

CH

R4

NH3 CH C

O

R1 R2

C

O

NH CH

R3

C

O

NH CH

R4

C+

+ ++

O

NH

NH

O

P1' P2'

S2 S1 S1' S2'

HN CH

H2O

P1P2

Reaction catalyzed by peptidases

scissile bond

Figure 2.1

NH3 CH C

O

NH

R1

CH

R2

C

O

NH CH

R3

C

O

NH CH

R4

COO-+

exopeptidase(carboxypeptidase)endopeptidase

exopeptidase(aminopeptidase)

Classifications of peptidases

Endopeptidases

• Representative example is -chymotrypsin• Regiospecifically hydrolyzes peptide bonds of

the aromatic acids• P1 -chymotrypsin is Phe, Tyr, and Trp• P1 for trypsin is Arg and Lys

NH3 CH C

O

NH

R1

CH

R2

C

O

NH CH

R3

C

O

NH CH

R4

COO+

P1

Scheme 2.2

EndopeptidaseR C

O

X

Ser195 O

H

NN H

His57

- O C

O

Asp102

C

O

XR

O

Ser

NN

H

His

H -O C

O

Asp

C

O

RO

Ser

His

H

HOH

- O C

O

NN Asp

C

OH

RO

O

Ser

NN

H

His

H -O C

O

Asp

R COOHSer195OH

NN H

His57

- O C

O

Asp102

+

++

+

acyl intermediate

+

acylation

deacylation

-XH

Mechanism for -chymotrypsin

showing catalytic triad

Figure 2.2

Evidence for Acyl Intermediate

NO2OCH3C

O

NO2O

initial burst phase

)

A400 nm

steady state phase

corresponds to 1 equivper equiv of enzyme

Time

-

(Release of

2.1

Reaction of chymotrypsin with p-nitrophenyl acetate: demonstration of an initial burst

Use of an alternate, poor substrate to change the rate-determining step

Scheme 2.3

Typical enzyme reaction in which the first step is fast

E•S'

E + P2slow

initial burst

fast

+ P1

E•SE + S

P1 = O NO2 P2 = CH3COO

For para-nitrophenylacetate

E•P2

Scheme 2.4

common acyl intermediate

Enzymatic rates - same

Nonenzymatic rates - different

PhCH CH C

O

OX

O

PhCH CH C

O

HOXO

2.2 2.3

+

Evidence for formation of an acyl intermediate

Reaction of -chymotrypsin with aryl cinnamate esters

14CH3C

O

O NO2

O

14CH3CO

O

O

14CH3C

O

O

2.5

2.4

2.6

O NO2

H2O

Scheme 2.5

To demonstrate covalent intermediate:

pH 5 pH 8

stops here

kinetically competent

Formation of an acyl intermediate in the reaction catalyzed by -chymotrypsin

below pH optimum for

catalysis

pH optimum

excess substrate

Fraction Number

RadioactivityAbs280

( ) ( )

Figure 2.3

Gel Filtration

(aromatic aminoacids in enzyme)

Scheme 2.6

reactivated enzyme

To support formation of acetylchymotrypsin

Reactivation of acetylchymotrypsin by hydroxylamine

14CH3CO

2.5

14CH3C

O

2.7

NHOHOH..

HONH2

O

Isolate and characterize

Rate of base hydrolysis of acetylchymotrypsin denatured by 8 M urea is identical to rate of base hydrolysis in 8 M urea with a model compound, O-acetylserinamide

H3C O

O

NH3+

O

NH2

Scheme 2.7

affinity labeling agent

O OP

O

F

OO

P OO

O

2.8

2.9

Reaction of -chymotrypsin with an organophosphofluoridate affinity labeling agent

To show involvement of a serine residue at the active site

Scheme 2.8

Affinity labeling agent

substrate protection

E•S

-S + S

E–IE•IE + I

Kinetics of affinity labeling of enzymes

• Irreversible inhibitors exhibit time-dependent inhibition

Reaction after E•I complex formation is rate limiting; therefore, time

dependent

Figure 2.4

Enzyme Inactivation

With [32P] get 1 equiv 32P bound to enzyme;

6 N HCl at 110 °C, 24 h gives [32P]phosphoserinePeptidase hydrolysis gives [32P]peptide containing modified Ser-195.

P

F

OOO

Correlation between loss of enzyme activity and incorporation of radioactivity during enzyme inactivation

loss of enzyme activity and incorporation of radioactivity correspond (1 : 1 inactivator : enzyme)

5000

0

100

0

% Enzyme Activity

Radioactivity(dpm)

Time

50 ( )( )

substrate inactivator (TPCK)

With [14C]TPCK get 1 equiv. [14C] bound; pepsin hydrolysis gives a [14C] peptide with His-57 modified

CH2 CH

NH

SO2

C

CH3

CH2 CH

NH

SO2

C

CH3

OCH3

O O

CH2Cl

2.11 2.12

Evidence for Histidine Participation

-chymotrypsin

(side reaction) (S)-N-Ac-L-Ala-L-Phe

(S)-N-Ac-L-Ala-L-Phe

Cl

CH3H

2.13

Mechanism of inactivation of -chymotrypsin by -chloromethyl ketones

OH

CH3

H

Evidence against a single SN2 reaction

Same stereochemistryas 2.13

No hydrolysis product in absence of enzyme(nonenzyme control)

Scheme 2.10

R

O

CH3R

Cl

O

O

O

R

OSer

SerSer O H

OH

R

O

OSer

Cl

HH

CH3 CH3H

H OH

B:

H

CH3R

O

CH3

OH

H

Ser OH

fast

195195 195

195

inversion

inversion

195

2.14 2.15

2.162.17

B:

Double inversion mechanism for inactivation of serine proteases by -chloromethyl ketones

Scheme 2.11

inversion of configuration

overall retention of configuration

Three possible mechanisms for inactivation

of -chymotrypsin by -chloromethyl ketones

N

HN

O

ClR

N

N

O

N

HN

O

N

HN

O

ClR

N

HN

Cl

R

O

N

N

R

O

O

O

N

HN

O

ClR

H

CH3

HCH3

HCH3

HCH3

HCH3

H

CH3

N

HN

Cl

R

O

O

HCH3

HH3C

N

HN

OO

H

H3C

R

R

N

HN

OH

O

H

H3C

R

R

EE:

E

E

O—H

E

-E

1)

2)

3) E

O—H

E

EE

2.18

2.19

OH OH

BO

B

HN H

Cl

CH3

O

OPh

AcNH

CH3

2.20

-Chymotrypsin was inactivated by 2.20, and X-ray crystal structure showed His-

57 alkylated with stereochemistry retained

acetyl-serine model

General base catalysis by imidazole solvent 2H isotope effect 2-3

C

O

OCH3 CH2 CH C

NH

C

CH3

O

2.21

O

NH2

Evidence for Deacylation Mechanism

Ph O

OHN N

Ph O

O

NH

N

2.22 2.23

Ser mimic His mimic

kH2O/kD2O = 3

Addition of PhCOO- as a model of Asp-102 increases rate 2500 fold

not active

Model study for deacylation step

Scheme 2.12

Improved model 1/18 rate of chymotrypsin

general base catalysis

Ph O

ON N H

O

OHO H

Ph O

OHN N

O

OHOH Ph

OH

O

HN N

O

OH

O

2.242.25

Chemical model for the deacylation step in -chymotrypsin

Table 2.1. Rate of Deacylation of Model Compounds Compared to Cinnamoyl-a-chymotrypsin

Compound Relative rate ( krel)

Ph O

O

chymotrypsin 1.0

2.22 2.6 x 10 -7

2.22plus benzoate ion

6.6 x 10 -4

2.24 5.6 x 10 -2

Ph O

OHN N

2.22

Ph O

ON N H

O

OHO H

2.24

Scheme 2.14

Aspartate Protease

Note: General acid-base catalysis, not covalent catalysis

Proposed mechanism for HIV-1 protease

NH

HO

N

OC

H

HO

O

Asp25

H

OH

O

Asp25'

O

NH

HO

N

O

H

H

O

O

Asp25

H

OH

O

Asp25'

O

NH

HO

N

O

H

H

O

O

Asp25

H

O

HO

Asp25'

O

N

HO

N

O

H

O

O

Asp25

H

O

O

Asp25'

O

H

H

NH

HO

N

O

H

O

O

Asp25

OH

O

Asp25'

O

H

H

O

R'

C

O

R'C

O

R'

C

O

R'C

O

R'

- -

+

-

-

δ

δ -δ

-δ..

+

--

..RR R

RR

Affinity labeling agent for CPA

labels Glu-270

CH2 CH COOH

NMe

CO

CH2Br

2.30

Carboxypeptidases (an exopeptidase)

Scheme 2.15

Zn++ is a cofactor

C NH

CHCOO

O

R

R

HOH

Glu270 COO-

Tyr248OH

Zn++

R O

O

Zn++

Tyr248-O

Glu270 COO

NH2 CH COO-

RH

HArg145+

General base catalytic mechanism for carboxypeptidase A

Scheme 2.16Not detected or trapped

C NH

CHCOO

O

R

R

C O-Glu270

O

Tyr248OH

Zn++ O

CR

O

COGlu270

Zn++

O

CR

Zn++

NH2 CH COO-

R

Glu270 COO-

O-

Arg145+

H2O

Nucleophilic mechanism for carboxypeptidase A

Principle of Microscopic Reversibility

For any reversible reaction, the mechanism inthe reverse direction must be identical to thatin the forward reaction (only reversed)

This can be a valuable approach to study enzyme mechanisms.

Scheme 2.17

R C

O

18O-

Glu CO

O-

R C

O

NH CHCO2-

R'

H2N CH

- H218O

CO2-

R'

Reverse of the general base mechanismReverse of general base catalytic reaction of carboxypeptidase A in the presence of H2

18O

Requires amino acid to release H2

18O

Scheme 2.18

Reverse of the nucleophilic mechanism

R C

O

18O-

Glu C

O

O-

R C

O

O C

O

GluR C

O

NH CHCO2-

R'H2N CH

CO2-

R'

- H218O

Reverse of nucleophilic catalytic reaction of carboxypeptidase A in the presence of H2

18O

Does not require amino acid to release H2

18O

Found amino acid is required for H218O release

(general base mechanism)

Scheme 2.19

From Crystal Structure of Ketone

Alternative mechanism for carboxypeptidase A on the basis of the X-ray structure with a ketone bound

270Glu O

O

H

O

Zn++

R

CHCOO-

:NH

O

R'

H3N127Arg

H

270Glu O

O

H

H

R

CHCOO-

:NH

C O-

R'H3N127Arg

O

Zn++

270Glu O

OR

CHCOO-

NH3+

O CO

R'Zn+++

+

tetrahedral intermediate

Functions of Zn++ Cofactor• Coordinate to H2O to make it more nucleophilic• Coordinate to carbonyl to make it more electrophilic

Scheme 2.20

R OR'

O

O H :B

H B

R

O

O HBR

O

O BHH OHB

OH :B

R'OH

RCO2H

H2O

Typical esterase mechanism

Covalent catalytic mechanism

OCH3

OHB

Me3NCH2CH2—O O

CH3

OB

H

H

Me3NCH2CH2—OH

B:

ester site

+-+

"anionic site"

Me3NCH2CH2OH + CH3COOH+

- +:B

H2O

Scheme 2.21

no anioncluster of aromatic residues instead(cation- complex)

Catalytic triad has a Glu instead of an Asp

Mechanism for acetylcholinesterase

Favored enantiomer substrate for lipases

Medium Large

H

2.31

R O

O

Scheme 2.22

O

O H

(1R,2S,5R)-menthyl pentanoate

+

O

O H

(1S,2R,5S)-menthyl pentanoate

lipase

HO H

(1R,2S,5R)-menthol

+

O

O H

(1S,2R,5S)-menthyl pentanoate

An example of the enantioselectivity of lipases/esterases

Useful for chiral resolutions of alcohols

Catalytic Antibodies (abzymes)

• Antibodies are proteins that scavenge macromolecular xenobiotics

• Form very tight complexes with macromolecule, which causes a cascade of events, leading to degradation of macromolecule

• A catalytic antibody is an antibody that catalyzes a chemical reaction

Construction of Catalytic Antibodies

• A transition state analogue that mimics the transition state of the desired reaction is synthesized--called a hapten

• Hapten is attached to a carrier molecule capable of eliciting an antibody response--called an antigen

• Antigen injected into a mouse or rabbit

• Monoclonal antibodies (ones that bind to one region of the antigen) are isolated for that antigen

• The monoclonals are tested for catalytic activity

Transition State Analogue Inhibitor

• Inhibitor molecules resembling the transition-state species should bind to enzyme much more tightly than the substrate

• Therefore, a potent enzyme inhibitor would be a stable compound whose structure resembles that of the substrate at a postulated transition state--a transition state inhibitor

Development of Catalytic Antibodies

Figure 2.5

R OR'

O

OHR

POR'

O

O

Ester hydrolysisintermediate

"Transition state" mimic

R OR'

O

HO

Comparison of an ester hydrolysis tetrahedral intermediate and a

phosphonate “transition state” mimic

Ph NH

PO

NHNH

OPh

O-

O O Me

O

NHX

O

O

2.32

mimics tetrahedral intermediate in ester hydrolysis

X = OH haptenX = macromolecule antigen (elicits antibody

response)

R1 = Bn R2 = HR1 = H R2 = Bn

NH2 O

NH

R1 R2

O

O

O

NH

Me

O

NH

NO2

2.33

Two different monoclonal antibodies raised, each catalyzes hydrolysis of different epimer

Aminations

Table 2.2. Types of Reactions Catalyzed by Glutamine-Dependent Enzymes

1)C OX C NH

2+

"NH3

"+

-

OX

2)

X

NH2

+ "NH3

"

3)C O

-

O

C NH2

O

"NH3

"

ATP

+

4)C

O

C

NH2

"NH3

"

ATP

+

Scheme 2.23

Glutaminase activity (generation of NH3)

• Free NH3 is toxic to cell - this protects cell from NH3

• NH3 can be substituted for Gln, but Km 102-103 higher

A covalent catalytic mechanism for the “glutaminase” activity of glutamine-dependent enzymes

NH2H3N

-OOCO

X

H B+

NH2

H3N

-OOC O

X

H:B

XH3N

-OOCOH B+

XGlu

Aminated product

+ "NH3"

acceptor

Scheme 2.24

Evidence for covalent catalysis

X

O O

NHOHXH

NH3+

-OOC-OOC

NH3+

2.352.34

NH2OH

Evidence for -glutamyl enzyme intermediate in glutamine-dependent enzyme

Figure 2.6

NH3+

OOCCl

O

NH3+

OOCNH2

O

2.36

Gln

Comparison of the structure of the -chloromethyl ketone of asparagine

with the structure of glutamine

irreversible inhibitor

substrate

modify Cys residue

Blocks enzyme reaction with Gln, but not with NH3; therefore 2 binding sites

2.37

O

CCH2 NH2I

N

O

O

Et

2.38

-OOCCH

+NH3

O

N N+ _

2.39

-OOCO CH

+NH3

O

N N

_+

2.40

Mechanism-based inactivators of Gln-dependent enzymes

Mechanism-based inactivator• Unreactive compound whose structure resembles the substrate (or product) for an enzyme• Acts like a substrate and is converted into a species that inactivates the enzyme• Cannot escape enzyme until it inactivates it

Scheme 2.26

partition ratio = 70 (d/c)

When R contains 3H, ratio of 14C/3H remains constant after inactivation

Mechanisms for inactivation of glutamine-dependent enzymes by -diazoketones

R 14CH

O

N N

H B+

R 14CH2

O

N N

X

R 14CH2

O

X

R 14CH2

O

N NX

XR X

O

R14CH2

O

XY

R 14CH2

O

YX

+ _+

ab

a

b

+

Glu or Ser PhCO214Me 14MeOH+

(E I) (E I')

a

2.39/2.40 2.41 2.42

2.432.44 2.45

c

cd

d

d

+ +

2.462.47

c

b

H2O14CH2N2

PhCO2H

-N2

-N2

H2O

Therefore, 2.39 is responsible for inactivation, not diazomethane (would only be 14C labeled)

Scheme 2.25

partition ratio = k3/k4

Ideally would be 0

k1

k-1

k3

k2 k4

E + I'

E • I' E - I''E • IE + I

Kinetics for mechanism-based inactivation

Acceptor reactions are mostly ATP-dependent

Scheme 2.27

An example where no ATP is required

5-phosphoribosyl-1-diphosphate amidotransferase

Amination reaction catalyzed by glutamine phosphoribosyldiphosphate amidotransferase

O

HO OHOP2O6

3-

=O3PO O

HO OH

NH2=O3PO

+ P2O74-

2.48-configuration β-configuration

+ ":NH3"

good leaving group

SN2-like reaction

What happens when NH3 is added to a carboxylic acid?

Scheme 2.28

+ PhCO2 NH4

+PhCO2H NH3

Function of ATP

Reaction of ammonia with benzoic acid

Scheme 2.29

ATP Chemical Equivalents

R Cl

O

R NH2

O

R NH2

O

O

O O

HO

O

R O

O O

+ +-SO2

+

2.49

+

2.50

SOCl2HCl

RCO2H

RCO2H-HCl

NH3

NH3-CH3COOH

Activation of carboxylic acid with thionyl chloride and acetic anhydride

ATP acts like SOCl2 or Ac2O

Figure 2.7

Requires Mg2+ for activity (coordinates to phosphate oxyanions)

Electrophilic sites on ATP

O

HO OH

N

O P

O

O

PO

O

O

PO

O

O

O

CH2 N

Nu-

β

-3 kcal/mol-7 kcal/mol

phosphoesterphosphoric acidanhydride

5'

ATP

N

N

NH2

Nu P

O

O

PO

O

O

O-

Nu P

O-

O

O

Nu P

O

O

PO

O

O

O Ado

Nu P

O

O

O Ado + PPi

or+ Pi

+ ADP

+ AMP

NuH + Pi

−

β−

−

NuH + PPiNuH + ADP

NuH + AMP

H2O

H2O

Figure 2.8

Products of reaction of nucleophiles at the -, β-, and -positions of ATP

Scheme 2.30

Asp COOH Gln C

O

NH2 Asn C

O

NH2 Glu COOH+

Mg•ATP Mg•AMP + PPi

+

Reaction Catalyzed by Asparagine Synthetase

Scheme 2.31

Two possible modes of attack to give AMP + PPi

Activation of aspartate by ATP followed by reaction with ammonia generated from glutamine

Asp C O

O C AMP

O

C PPi

O

Asp

Asp

PPi+

+

.

PPi

+

or Asn + AMP +

-attack

β-attack

ATPMg

AMP

NH3

Gln

-Glu

Scheme 2.32

[18O] AMP

[18O] PPi

*experimental result

Use of 18O-labeled aspartate to differentiate attack at the - or β-positions of ATP

AspC18O

O

AspC 18O

O

-O P O P O P O Ado

O

O-

O

O-

O

O-

-O P O P O P O Ado

O

O-

O

O-

O

O-

AspC 18O

O

P OAdo

O

O-

AspC 18O

O

P O P O-

O

O-

O

O-

C

O

-18O P O P O-

O

O-

O

O-

Mg++

Mg++

-18O P OAdo

O

O-

Asn NH2

C

O

Asn NH2β-attack

-PPi

-AMP

-attack

+

+

NH3

NH3

*

Scheme 2.33FGAR

Reaction catalyzed by formylglycinamide ribonucleotide (FGAR) aminotransferase

Important enzyme in purine biosynthesis

O

HO OH

NH=O3PO

O

HNOHC

O

HO OH

NH=O3PO

NH

HNOHC

2.52

+ Mg•ADP+ Gln + Mg•ATP

2.51

+ Pi + Glu

Scheme 2.34

Use of 18O-labeled FGAR to differentiate attack at the - or β-positions of ATP

-O P

O

O-

PO

O

O-

O

O

HO OH

NH=O3PO

NH2

HNOHC

P Ado

Mg++

NH

OHCN

R

18O

H

NH

HN

R

18O

OHC

P

O-

O

O-

18O P

O-

O

O-

NH

: NH2HNOHC

R

18O P

O

O

O

ADP

+

O

O-

Gln

-Glu

:NH3

Scheme 2.35

Partial exchange reaction - a way to detect intermediates in multi-step reactions

Therefore attack occurs at the -position

Use of AD32P in a partial reaction to test for reversibility of FGAR aminotransferase and test whether ADP or Pi is

released during the reaction (Gln omitted)

-O P

O

O-

PO

O

O-

O P

Mg++

NH

OHCN

R

O

H

NH

HN

R

O

OHC

P

O-

O

O-

O

O

OAdo

32PO

O

O

O P

Mg++

O

O

OAdo

-O P

O

O-

32PO

O

O-

O P

Mg++

O

O

OAdo

NH

OHCN

R

O

H

NH

HN

R

O

OHC

P

O-

O

O-

2.53(ATP)

2.53

+

+

(AD32P)

(AT32P)

+

ADP

Forwardreaction

Reversereaction

Scheme 2.36

If β-attack had occurred:

partial exchange w/ 32Pi

-O P

O

O-

PO

O

O-

O P

Mg++

NH

OHCN

R

O

H

NH

HN

R

O

OHC

P

O-

O

O

O

O

OAdoP OAdo

O

O-+

(ATP)

Pi

Pi

Pi

Outcome if FGAR aminotransferase proceeded by formation of ADP phosphate ester

No AT32P would have been formed with added AD32P because ADP would not be an intermediate

If neither experiment leads to incorporation of 32P into the ATP, it does not mean that neither intermediate is formed

• Assumed enzyme followed an ordered mechanism and that the first partial reaction could proceed in the absence of glutamine: Maybe enzyme needs the glutamine to be bound before activation occurs Binding of glutamine may cause a conformational change that sets up binding site for FGAR and ATP

• Another potential problem - ADP generated in the first partial reaction may bind very tightly, so dissociation and exchange with AD32P do not occur

Aspartate as the NH3 source

Scheme 2.37

-attack

Mechanisms for the reactions of argininosuccinate synthetase, an aspartate-dependent enzyme, and argininosuccinate lyase.

ATP is abbreviated as POPOPOAdo :NH2

C 18O

NH

CH2

CH2

CH-OOC

NH3+

NH3 CH

CH2

COO-

COO- NH2+

NH2

NH

CH2

CH2

CH-OOC

NH3+

COO-

-OOC

NH2

C 18OPOAdo

NH

COO-

NH3+

NH2 C

H

CH2COO-

COO-

:B Enz

NH2+

NH

NH

NH3+-OOC

CH

COO-

CHCOO-

H

(argininosuccinate lyase)

1. argininosuccinate synthetase +

2.55

Mg•AMP + PPi+

++

PPi

POPO-POAdo

Mg•ATP

2.54 2.56

2.572. argininosuccinate lyase

(argininosuccinate synthetase)

AMP(18O)

(18O)

Figure 2.9

Phosphorylations

R O P

O

O-

O-

X PO32- Y PO3

2-

R O P

O

OR'

O-

H2O ROH + Pi

+ X-

H2O ROPO32- + R'OH

phosphatase

phosphodiesterase

kinase

electrophile nucleophile enzyme family reaction type

+

+

+

products

transfer

hydrolysis

hydrolysis

Y-

Comparison of the reactions of a phosphatase, a phosphodiesterase, and a kinase

Scheme 2.38

metaphosphate

R O P

O

O-

O-

B+ H

HO H

:B

Enz X EnzX P

O

O-

O-

R O P

O

O-

O-

B+ H

R OPO32-

HO H

:B

R O P

O

O-

O-

B+ H

HO H

:B

P

OO

O-

HO H:B

R O P

O

O-

O-

B+ H

P

OO

O-

EnzX

EnzX P

O

O-

O-

ROH + Pi

+ ROH

Enz-X + Pi

HO H

:B

+ General Acid-Base Catalysis-associative

Covalent Catalysisassociative+

ROH + Pi

R O P

O-

O-O-

PiROH +General Acid-Base Catalysis-dissociative

ROH +

B+ H

Enz-X + Pi

Covalent Catalysisdissociative

O1)H

2)

H

1)

B

2)

‡

SN2

A

B

C

Three general mechanisms for phosphatases

Phosphatases

How would you test mechanism?• Mechanism C differentiated from mechanisms A and B

by incubation with H218O

• Associative and dissociative mechanisms are differentiated

by secondary kinetic isotope effects:

Substitution of the phosphate oxygen atoms with 18O gives slower reaction in an associative mechanism (lower bond order; 18O-P is stronger than O-P bond; normal secondary isotope effect), but a faster reaction in a dissociative mechanism (18O=P is higher bond order; more stable transition state; lower activation energy; inverse secondary isotope effect)

•Associative mechanism gives inversion of stereochemistry

about the phosphorus atom, but this may or may not occur

with a dissociative mechanism

Scheme 2.39

H218O adds to P

2.58 + [14C]2.59 [14C]2.58

2.58 + 32Pi No [32P]2.58

[32P]2.58 [32P]peptide

G 6-P’ase

phenol tryptic

quench digestion

KOH[32P]His

G 6-P’ase

G 6-P’ase

digestion

Therefore phosphoenzyme formed reversibly with release of glucose followed by irreversible hydrolysis of phosphoenzyme to Pi

Reaction catalyzed by glucose 6-phosphatase

O OHOH

OH

HO

O P

O

O

O

O OHOH

OH

HO

OH

+ H2O + Pi

2.58 2.59

(excludes SN2)

Reversible reaction

Irreversible Pi formation

Scheme 2.40

Common Mechanistic Feature (partial reaction) of the Enolase Superfamily

Common active site structural feature to catalyze a variety of different reactions in different enzymes.

R O

O-R' H

B:

R O-

O-R'

1,1-proton transfer (racemization)

β-elimination of OH-

β-elimination of NH3

β-elimination of R"COO-

M2+ M2+

Superfamilies of Enzymes

Scheme 2.41

Dissociative covalent catalytic mechanism for VH1 dual-specific Tyr phosphatase

(also hydrolyzes phosphoserine and phosphothreonine residues)

pKa 5.6

Expected stereochemistry of phosphate?

Mechanism for the reaction catalyzed by human dual-specific (vaccinia H1-related)

protein tyrosine phosphatase

92Asp

OOH

O P

O

O-

O-124Cys-S

OH

92Asp

OO-

124CysSP

O

O-

O-

H

O

H

92Asp

OOH

124Cys-SP

O

OO-

92Asp

OO-

HPO4-2

Figure 2.10

Associative mechanism - favored by metal ions

Ser/Thr phosphatase PP1

Metal ions make the H2O more nucleophilic and the phosphate more electrophilic

Stereochemistry?

(a) Molecular model of the active site of protein serine/ threonine phosphatase PP1 with tungstate ion (WO4) bound; (b) Schematic of the catalytic mechanism based on the crystal structure and kinetic studies

R

O

P O-O

O

CH2

O

OO

C

P O-O

O

CH2

O

OHO

A

P O-O

OR'

B+ H

:B

O

OO

C

PO O-

O

OHO

AHO

P

B:H OH

B+H

H

-O O

OR'

R

O

P O-O

O

CH2

O

OH

C

R

O

P O-O

O

CH2

+

2.62

2-O3PO

Scheme 2.42

Phosphodiesterases

12His

119His

General acid/base-catalyzed reaction for ribonuclease A

Kinases

• Transfer the -phosphoryl group of nucleoside triphosphates (originally only ATP) to an acceptor

• Now generalized to reactions at the -, β-, or -position of any nucleoside triphosphate

Kinases

Scheme 2.44 phosphoenolpyruvatePEP

trapped w/Br2

No evidence for a phosphoenzyme intermediate

In the presence of an ATP mimic in 3H2O, 3H is incorporated into pyruvate

H2C

H

C

O

COO- CH2 C

O

COO- CH2

OPO3=

COO-

2.68

+ ADPP-O-P-O-P-O-Ado

2.66 2.67

HB: B:

Mechanism for pyruvate kinase (ATP is abbreviated POPOPOAdo)

Scheme 2.45

CH3C

O

O CH3C

O

OPOAdo CH3C

O

SCoA P-O-P-O-P-O-Ado

PPi

+ AMP

N

N N

N

O

HO OPO3=

CH2 OP

O

O-

OP

O

O-

OCH2 C

CH3

CH3

C

OH

H

C

O

NH CH2 CH2 C

O

NHCH2CH2SH

NH2

2.69

+ CoASH

CoASH

Mechanism for acetyl-CoA synthetase (ATP is abbreviated POPOPOAdo)