The Organic Chemistry of Enzyme-Catalyzed Reactions

Chapter 9

Isomerizations

Conversion of one molecule into another with the same formula

• Hydrogen shifts to the same carbon: [1,1]-H shift

• Hydrogen shifts to the adjacent carbon: [1,2]-H shift• Hydrogen shifts to two carbon atoms away: [1,3]-H

shift

Isomerizations

Not PLP - no visible absorbance

Not pyruvoyl - acid hydrolysis gave no pyruvate

No M2+ - EDTA has no effect

No acyl intermediates - no 18O wash out of [C18O2H]Glu

Not oxidation/reduction - 2H is incorporated into C-2 from 2H2O

Therefore deprotonation/reprotonation mechanism

[1,1]-Hydrogen Shift

Racemase with no cofactors

Glutamate racemase

Scheme 9.1

[1,1]-Hydrogen ShiftAmino acid racemases

One base: substrate proton transferred to product

(A) One-base mechanism for racemization (epimerization), (B) Two-base mechanism for racemization (epimerization)

R

H

NH3+

COO-

B

R

NH3+

-OOC

B

R+H3N

-OOC

B

H

R

H

NH3+

COO-

B BH B B

R

NH3+

-OOC

H

R+H3N

-OOCH

BH B

Ha

ab

H

b

A

B

also, primary kinetic isotope effect with [2-2H]GluWith Glu racemase: solvent deuterium in product, not substrate

Two base: incorporated proton from solvent(B)

Figure 9.1

in D2O

An “Overshoot” Experiment with (R)-(-)-glutamate to Test for a Two-base Mechanism for Glutamate Racemase

0

20

60

100

-20

-60

-100

Time (sec)1000 2000 3000

Scheme 9.2

Another Test for a Two-Base MechanismElimination of HCl from threo-3-chloroglutamic acid by the C73A and C184A mutants for glutamate racemase

COO-

COO-

Cl

NH3+

H

H

COO-

-OOC

Cl

NH3+

H

H

SS

COO-

COO-

Cl

NH3+

H

H

COO-

-OOC NH3+

HCOO-

COO-+H3N

H

COO-

-OOC

Cl

NH3+

H

H

C184A

COO-

-OOC O

HD

COO-

COO-O

H

C73A

9.1

D

9.2 9.2

S RS R

SS

D2O

RR

D2O

C73C184

Scheme 9.3

Inactivation by ICH2COO- only after a reducing agent is added (RSH or NaBH4)

Proposed Mechanism for Proline Racemase

HN

H HN

S

HOOC

S

H S

S

S

SH

HN

H

S

S

H

9.3

HO

HOO

HO

‡δ

HN

O

HOδ

H

H

Reduces active site disulfide to dithiol

Transition State Analogue Inhibitor

HN-OOC

9.4

Because substrates bind tightest at the transition state of the reaction, a compound resembling the TS‡ structure would be more tightly bound

TS‡ analogue inhibitor for Pro racemase

resembles 9.3

Scheme 9.4

Pyridoxal 5-Phosphate (PLP) Dependent Racemases

NH

NH

CCH3

H

COO-

NH

NH

CCOO-

CH3

NH

NH

CCH3

COO-

H

CH3

OH OH

CH3

OH

CH3

:B

=O3PO

+Keq ~ 1

L-Ala D-Ala

=O3PO =O3PO

+

+

NH3

CCH3

H

COO-

+ +

+

PLP

+NH3

CCH3

COO-

H

BH

quinonoid intermediate

PLP

PLP

Proposed mechanism for PLP-dependent alanine racemase

Usually, a one-base mechanism

How can PLP enzymes catalyze selective bond cleavage?

PLP was a coenzyme for decarboxylases (break C-COOH bond) and now for racemases (break C-H bond)

Stereochemical Relationship Between the -Bonds Attached to C and the p-Orbitals of the -System

in a PLP-Amino Acid Schiff Base

Figure 9.2

NH

NH

H

R

-OOC

The -bond that is parallel to (overlapping with) the p-orbitals will break (C-H in this case)

PLPall sp2 + p atoms

Figure 9.3

Dunathan Hypothesis for PLP Activation of the Bonds Attached to C in a PLP-Amino Acid Schiff Base

C

RH

COO-

C

H-OOC

R

C N CH

H

N CHN CH

R COO-

+

+

BA C

pyridine ring of PLP

The -charge stops free rotation, which results in selective bond cleavage

The rectangles represent the plane of the pyridine ring of the PLP. The angle of viewing is that shown by the eye in Figure 9.2.

Scheme 9.5

No internal return in either direction

Other Racemases

Ph CO2-

HO H

Ph CO2-

H OH

S-mandelateR-mandelate

Reaction catalyzed by mandelate racemase

With (R)-mandelate no -H exchange with solventWith (S)-mandelate there is exchange with solvent

Scheme 9.6

solvent exchange

no solvent exchange

H297N mutant is capable of exchanging the -H of the S-isomer, but not the R-isomer

A Two-base Mechanism for Mandelate Racemase that Accounts for the Deuterium Solvent Exchange Results.

166Lys ND2

Ph

HOH

166Lys N

H

D

D

PhO-

OH

OO-

O

Ph

HO H

ND

N

297HisND

N

297His

H

PhO-

OH

O

O-

OPh

HO H

O-

O

Ph

DOH

O-

O

+

Mg2+

Mg2=

Mg2+ Mg2+

(S)

(R)

A

B

Lys-166 acts on the (S)-isomer, and His-297 acts on the (R)-isomer

Scheme 9.7

H297N Mutant Capable of Elimination of HBr from (S)-9.5, but not from the (R)-isomer

K166R mutant catalyzes elimination of HBr from the (R)-isomer, but not from the (S)-isomer

Elimination of HBr from (S)-p-(bromomethyl)mandelate, catalyzed by the H297N mutant of mandelate racemase

Br

NH2Lys-166

COO-

OH

COO-

O

9.5 9.6

COO-

OHH

-HBr

EpimerasesPeptide epimerases

Scheme 9.8

Mechanism 1

With 18O in the Ser OH group, no loss of 18O as H218O

Elimination/addition (dehydration-hydration) mechanism for peptide epimerization

NH

OH

O

Phe-Ala-OH

O

AcNH-Gly-Leu

H

:B

NH

O

Phe-Ala-OH

O

AcNH-Gly-Leu

NH

OH

O

Phe-Ala-OH

O

AcNH-Gly-Leu

H

BH

BH

NH

OH

O

Phe-Ala-OH

O

AcNH-Gly-Leu

H:B

BH

NH

OH

O

Phe-Ala-OH

O

AcNH-Gly-Leu

H:B

BH

9.7

BH

-OH

Therefore, mechanism 1 is unlikely.

Scheme 9.9

Mechanism 2

10 mM NH2OH has no effect on product formation

NH

O

O

Phe-Ala-OH

O

AcNH-Gly-Leu

H

:B

NH

OH

O

Phe-Ala-OH

O

AcNH-Gly-Leu

H

BH

NH

O

Phe-Ala-OH

O

AcNH-Gly-Leu

H:B

B

H

HH

H

O

9.8

-Cleavage Mechanism for Peptide Epimerization

Therefore, mechanism 2 is unlikely.

Scheme 9.10

Mechanism 3

In D2O D is incorporated into product, not substrate (short incubation; monitored by electrospray ionization mass spectrometry)

NH

OH

O

Phe-Ala-OH

O

AcNH-Gly-Leu

H

B

NH

HO

O

Phe-Ala-OH

O

AcNH-Gly-Leu

H

BH

NH

OH

O

Phe-Ala-OH

O

AcNH-Gly-Leu

H:B

BH

BH

BH

BH

B:

Deprotonation/Reprotonation Mechanism

Deuterium isotope effect for [-2H]-peptides in the L- to D-direction is different from that in the D- to L-direction (two-base mechanism)

These results are consistent with mechanism 3.

OHO

H

CH3

H

OdTDPOH

HH

OHH

B+ H O

O

H

CH3

H

OdTDPOH

HH

OH

N

H H

NH2

O

R

9.9 9.10

NADPH

epimerase reductase

O

O

CH3

HH

OdTDPOH

HH

OHB+ H

:B

B+ H

B:

OCH3

H

H

OdTDPOH

HOH

H

O

OO

CH3

HH

OdTDPOH

HH

OH

B:

B+ HO

O

H3C

H

H

OdTDPOH

HH

OH

B+ H

B:

Scheme 9.11

Epimerization with Redox Catalysis

two different enzymes

C-H cleavage at C-3 and C-5 show kinetic isotope effects (3.4 and 2.0, respectively)

Proposed mechanism for dTDP-L-rhamnose synthase-catalyzed conversion of dTDP-4-keto-6-deoxy-D-glucose (9.9) to dTDP-L-rhamnose (9.10)

In 2H2O 2H incorporation at both C-3 and C-5

Partial exchange gives only C-3 proton exchange, never only C-5 proton exchange (ordered sequential mechanism)

UDP-Glucose 4-Epimerase

UDP-glucose UDP-galactose

No change in oxidation state, but is deprotonation/reprotonation reasonable?

OOH

OH

O UDPOH

OH

OOH

OH

O UDPOH

HO

9.11 9.12

In H218O, no incorporation of 18O into product

Scheme 9.12

The enzyme requires NAD+; no exchange with solvent

reverse reaction

without OH

proposed intermediate

Tritium is incorporated from NAD3H into a derivative of the suspected intermediate of the UDP-glucose 4-epimerase-catalyzed reaction

OOH

CH3

O dTDPOH

OO

OH

CH3

O dTDPOH

HO

3H

9.13

E•NAD3H +

Scheme 9.13

Evidence for 9.14: incubate enzyme with UDP-galactose,quench with NaB3H4. 3H at C-4 of both UDP-glucose and UDP-galactose

Proposed Mechanism for Reaction Catalyzed by UDP-Glucose 4-Epimerase

OOH

OH

O UDPOHO

H OOH

OH

O UDPOH

O

B HO

OH

OH

O UDPOH

H

HO

NAD H

+

9.14+

H:B

NAD

NAD

Scheme 9.14

Mechanism to Account for Transfer of Hydrogen from the Top Face of UDP-glucose and Delivery to the Bottom Face of the 4-Keto Intermediate

OOH

OH

H

O OH

OBH

O

OHOHO

O OH

O

OHO

HO

OH OH

HUDP

UDP UDP

N

H2N O

R

H

:B

N

H2N O

RH

H

-NAD+

Scheme 9.15

No change in oxidation state, but NAD+ required

Mechanistic Pathway for the GDP-D-mannose-3,5-epimerase-catalyzed Conversion of GDP-D-mannose (9.15) to GDP-L-galactose (9.18)

OOH

OH

O GDP

OH

OH

H OOH

OH

O GDP

OHO

O

OH

OH

O GDP

OH

O

OH

O GDP

OHO

9.15OH9.16

O

OH

OH

O GDP

OH

OH

9.179.18

O

H NAD+

NAD+ NADH

NADH

[1,2]-H Shift

Scheme 9.16

Lobry de Brun-Alberda von Ekenstein Reaction

Reaction catalyzed by aldose-ketose isomerases

CHO

C OHH

R

CH2OH

C O

R

9.209.19

Scheme 9.17

Two

Mechanisms

suprafacial transfer of H

cis-Enediol mechanism for aldose-ketose isomerases

C

C O

R

H

OHHBC

C

OH

OR

H

BB:

H

C O

C

R

OHH

B HH

H

:B BH

R

OHHOH

B:

BHOR

OHHB H

H

:B

9.22

OR

*

H

HR OH

*

*

9.21

B

**

B

*

H

(2R)

(2R)

re

re

pro-Rcis-enediol

Mechanism 1

Scheme 9.18

Partial incorporation of solvent observed - inconsistent with hydride mechanism

Hydride transfer mechanism for aldose-ketose isomerases

BH

:BC

C O

R

H

O

H

:B

BH

*

C

C O

R

H

OH

H*

H

Mechanism 2

Scheme 9.19

[1,3]-H Shift

Enolization

removes pro-R hydrogen

Reaction catalyzed by phenylpyruvate tautomerase

CO2-

O

HSHR

R R

HS

OH

CO2-

R = H or OH

Scheme 9.20

Two Conformers PossibleConformations of phenylpyruvate that would form Z- and E-enols by phenylpyruvate tautomerase

CO2-

O

HS HR

H B

B:

CO2-

OH

O

CO2-

HS HR

H B

B:

CO2-

OH

anti

Z

E

syn

favored inhibitors

Therefore syn geometry to E enol most likely

To Test for Favored Conformation

F

CO2-

R CO2-

F

R

CO2-

RCO2

-R

9.23 9.259.24 9.26

Scheme 9.21

Allylic Isomerizations

Carbanion mechanism for allylic isomerases

B:H

B HH

This H could come from the substrate (if no solvent exchange)

Mechanism 1

Scheme 9.22 This H comes from solvent, not from the substrate

Carbocation mechanism for allylic isomerases

B+H

H HH H

B:

Mechanism 2

Scheme 9.23

Unlikely -- [1,3]-hydride shift is allowed antarafacial,which is geometrically impossible

[1,3]-Sigmatropic hydride shift mechanism for allylic isomerases

H H

Mechanism 3

Scheme 9.24

Carbanion Mechanism

Principal reaction transfers 4-H to 6-position; therefore suprafacial

Reaction catalyzed by 3-oxo-5-steroid isomerase

O

H

O

H H

O

H

OHH

12

34

5 6

9.27 9.28(D)

(D)

Eliminates carbocation mechanism and [1,3] hydride shift

Scheme 9.26

Evidence for an Enol Intermediate in the Reaction Catalyzed by 3-Oxo-5-steroid Isomerase

O

O

O

O

O

O

O

HO

9.329.31

9.31

10%

nonenzymaticpH 4.5

9.32

9.349.33(90%)

+

enzymatic

enzymatic

Scheme 9.27

Kinetic Competence of Enol

same rates

Further evidence for an enol intermediate in the reaction catalyzed by 3-oxo-5-steroid isomerase

O

HO

O

O

O

O9.35 9.36 9.37

from NOE studies

From Site-directed Mutagenesis, Tyr-14 is the Acid and Asp-38 the Base

O

H

H

Asp-38

O O

OH Tyr-14

OH Tyr-14

OH Tyr-14

9.38

suprafacial

orthogonal (favored)

antarafacial

To probe the function of Tyr-14

Scheme 9.28

Uv spectrum bound to enzyme is same as neutral amine.

Structure bound to enzyme even at low pH (pKa of the phenol must be very low).

Reactions Designed to Investigate the Function of Tyr-14 at the Active Site of 3-oxo-5-steroid Isomerase

H2N

OH

H3N

OH

HO

O

-O

O

9.39

9.40

- H+

+ H+

+

Therefore Tyr-14 does not protonate C-3 carbonyl

Therefore Tyr-14 H bonds to dienolate

Scheme 9.29

Carbanion Mechanism

Mechanism for suprafacial transfer of the 4-proton to the 6-proton of steroids catalyzed by 3-oxo-5-steroid isomerase

O

O

O

O

O

O

H2H HOH

Tyr-14

COO-

Asp-38

O

Tyr-14

HCOO

Asp-38

2HO

H

Tyr-14

2H H

COO-

Asp-38

_

Asp-99 Located Adjacent to Tyr-14

Scheme 9.30

One mechanism for the function of Asp-99 in the active site of 3-oxo-5-steroid isomerase

O

H H

O

HOTyr14

HCOOAsp99

O

OH

O

HOTyr14

HCOOAsp99

OH

O

HOTyr14

HCOOAsp99

OO

38Asp

H H

38Asp

O OO

38Asp

H

equilenin

Crystal structure with equilenin bound is consistent with Asp-99 and Tyr-14 both coordinated to oxyanion

HO

O

9.41

4-Oxalocrotonate Tautomerase

O

CO2-

CO2-

OH

CO2-

CO2-

O

CO2-

CO2-

9.42 9.43 9.44

Scheme 9.32

From deuterated substrates, substrate analogues, and reactions run in D2O, 9.42 to 9.44 is suprafacial(one-base mechanism)

Scheme 9.33

Carbocation Mechanism

No exchange of solvent into substrate, only into product

Reaction catalyzed by isopentenyl diphosphate isomerase

P

O

O-

P

O

O-

O-

9.469.45

OO P

O

O-

P

O

O-

O-OOMg++

isopentenyl diphosphate dimethylallyl diphosphate

One base mechanism

rate is 1.8 10-6 times Ki = 14 pMOP2O6

3-

CF3 OP2O63-

HN+

OP2O63-

9.48 9.49

Evidence for a Carbocation Mechanism

transition state analogue inhibitor

Scheme 9.35

Proposed Mechanism for Isopentenyl Diphosphate Isomerase

OPP

B 2H

OPP

H

B:

OPP2H

2H

Scheme 9.36

Aza-allylic Isomerization

+NH

H

+NH

H

Scheme 9.37

PLP-dependent

AminotransferaseReaction catalyzed by L-aspartate aminotransferase

-OOC CH214C

H

COO-

NH3+

13C COO-

O

-OOCCH214C

18O

COO- CH313C

H

COO-

15NH3+

15

+CH3+H2

18O

Scheme 9.38PMP

First Half Reaction Catalyzed by Aspartate Aminotransferase

NH

NH

OH-OOC 14C COO-

H

15NH2

-OOC 14C COO-

H

15NH

NH

OH

B:

-OOC 14C COO-

15NH

NH

OH

B H

-OOC 14C COO-

15NH

NH

OH

-OOCCH214C

18O

COO-

15NH3

NH

OH

9.50

aldimine

=O3PO=O3PO

=O3PO

=O3PO

=O3POslow step

quinonoid

9.51

9.52

..

H218O

see Scheme 8.39

Scheme 9.39

Second Half Reaction Catalyzed by Aspartate Aminotransferase

CH3 13CCOO-

15NH

NH

OH

B15NH3

NH

OH13C COO-

O

CH3 HB:

CH3 13CCOO-

15NH

NH

OH

HCH3 13C

COO-

15NH

NH

OH

H

NH2

NH

NH

OH

CH3 13CCOO-

15NH3

H

=O3PO

=O3PO=O3PO

=O3PO

=O3PO

9.53

9.52

This is the reverse of the mechanism in Scheme 9.38

Crystal structures of:

• native enzyme with PLP bound

• substrate reduced onto PLP

• enzyme with PMP bound

All are consistent with mechanisms in Schemes 8.39 and 9.38

pseudosubstrate quinonoid form observed at 490 nm

NH

OH

NH

COO--OOC

OH

NH3

COO--OOC

OH

9.54

+

+

=O3PO

9.55

Evidence for Quinonoid Intermediate

Scheme 9.40

-H is transferred to the CH2 of PMP suprafacially; therefore one-base mechanism-2H removed from si-face and delivered to pro-S CH2 of PMP

Stereochemistry of Proton Transfer in the First Step Catalyzed by Many PLP-dependent Aminotransferases

N

-O

H

B:

N

2H

H

H3C OPO3=

H

COO-

R

N

-O

H

B

N

2H

H

H3C OPO3=

H

-OOC R

N

-O

H

B:

N2H

HH3C OPO3

=

H

-OOC R

N

-O

H

H2N2H

H

H3C OPO3=

-OOC R

O

9.56

9.57

H2O

pro-S

Scheme 9.41

Cis-Trans Isomerization

GSH acts as a coenzyme, not as a reducing agent

No 2H incorporated into substrate or product from 2H2O

Reaction catalyzed by maleylacetoacetate isomerase

COO-

COO-

O O

COO-

O O

-OOCGSH

9.58 9.59

Scheme 9.42

Proposed Mechanism for the Reaction Catalyzed by Maleylacetoacetate Isomerase

COO-

COO-

O O

COO-

O O

-OOCCOO-

COO-

O O

GS SG

COO-

O O

-OOCGS

Scheme 9.45

Phosphate Isomerization

only -anomer binds

Reaction catalyzed by phosphoglucomutases

O

OHOH

HO

O

OPO3=

HO

OPO3=OH

HO

OH

HO

9.679.66

Scheme 9.46

Native State of Enzyme is Phosphorylated

tightly bound

Shown as associative, but could be dissociative

Proposed mechanism for the reaction catalyzed by phosphoglucomutases

O

OHOHHO

HOO

OPO3=OHHO

O

HO

P

O

O-O O-Ser

B H

Ser O PO3=Ser O

PO-

O

O-

O

OOHHO

O32PO3=

HO

B H

O32PO3=

B32 H

H:B

9.689.67 9.66

Overall retention of configuration at phosphateDouble inversion

Scheme 9.47

Model Reaction for a Dissociative Mechanism of Phosphomutases

NO2

OP

O

18O-S-

OH

NO2

OO

NO2

OHO

PO

18O-S

solventcage

~ 40% retention

PO

18O-S

H