Vitamin B12 Mechanism

Wan-Chun Chung 4/18/2011

2

Outline

Introduction

Isomerase

mechanism

problems

Methyltranferase

mechanism

problems

Dehalogenase

mechanism

3

Introduction

Brain, nervous system, blood formation,

metabolism, DNA synthesis

Antipernicious anemia factor

First water stable organometallic complex

Cofactor of

1.adenosylcobalamin-dependent isomerases

2.methylcobalamin-dependent methyltransferases

3.dehalogenases

Holoenzyme : apoenzyme+cofactor

4

Structure

NN

N N

Co

R

CH3

CH3

H2NOC

H2NOC

H3C

H3C

H

H2NOC

CH3

NH

H

CH3O

P

OO

O

OH

N

N CH3

CH3

CONH2

CH3

CH3

CONH2

CH3

CONH2

O

HO

Corrin ring

Dimethylbenzimidazole

Base-on/base-off

nucleotide

R= deoxyadenosyl

CN-, OH-, H2O

N

N

N

N NH2

O

OHHO

CH2

cobalamin

5

Three main types Isomerase

Diol dehydrase, ammonia lyase, glutamate mutase, methylmalonyl CoA mutase, ribonucleotide reductase

Methyltransferase

Methionine synthase, acetyl-CoA synthase

Reductive dehalogenase

anaerobic dehalogenase

R Cl 2e- 2H+ RH HCl

C C Z

HX

C C Z

XH

R CH3 R' H R H R' CH3

Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247

6

Isomerase

C C Z

HX

C C Z

XH

Class I : Base-off/His-on

Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247

7

Homolysis v.s. Heterolysis

C

Co

B

C

Co

B

C

Co

B

CoIII

d6, l.s.

18 e-

CoII

d7, l.s.

17 e-

CoI

d8, l.s.

16 e-

CoII EPR evidence ( also substrate radical)

Some reactions have inversion while some others have retention on substrate no planar intermediate

Diol dehydrase : H(OH)CCH(=O) very stable, deactivates cycle

CoIII CH2AdoCoII

CH2Ado

CoII

CH3Ado

R

CoIII CH2AdoCoI

CH2Ado

CoI

CH3Ado

R

RH

RH

Homolytic pathway !

Jordan, R. B. Reaction Mechanisms of Inorganic and Organometallic Systems;

Oxford, 2007

8

Important role of protein:

constrains and protects reactive intermediate (.R), inhibits unwanted side reactions

Base-off/his-on

No .CH2Ado observed it’s reactive and short-lived

k>300 s-1 stopped-flow

KIE=30 not only Co-C cleavage but coupled with hydrogen abstraction is RDS

Jordan, R. B. Reaction Mechanisms of Inorganic and Organometallic Systems;

Oxford, 2007

9

Isomerase Class II : Base-on

Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247

10

Problems Thermal stability of Co-C bond compare to

high reactivity of enzymic reaction

1.

2. L(DMGH)2Co-CH3 stretching frequency, L affects the stability of CoII

N

N

O

O

N

OH

HO

N

Co

(DH)2Co (or

(DMGH)2Co)

Halpern, J.; Ng, F.T.T; Rempe, G. L. J Am Chem Soc,1979, 105, 7124

Jordan, R. B. Reaction Mechanisms of Inorganic and Organometallic Systems; Oxford,, 2007

11

In water

heterolysis takes place

Finke & Hay found ΔH*=33±2 kcal/mol

ΔS*= 11±3 cal/mol*K k=1*10-9 s-1

(cp. k>300 s-1)

enzyme distorts the coenzyme so that the

homolysis is 15 kcal/mol more favored

Hay, B. P.; Finke.R. G. J. Am. Chem. Soc. 1986, 108, 4820

12

Problems Limited precedent for radical rearrangement

Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247

13

Methyltransferase Methionine synthase

homocysteine

HN

N NH

N

CH3

R

O

H2NHS

O

O-

H3N H

HN

N NH

N

H

R

O

H2NH3CS

O

O-

H3N H

CH3-H4folate H4folate methionine

CH3

CoIII

B

-S

O

-O

NH3

H

CoI

S

O

-O

NH3

H

CH3

H+

HN

N NH

N

H

R

O

H2N

HN

N NH

N

CH3

R

O

H2N

Shriver textbook

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Nucleophile attack vs Oxidative addition

Oxidative addition needs two cis vacant site impossible

For overall reaction, the methyl configuration is

retention both steps retention or inversion

Absence of EPR signal for CoII or organic radical

no homolytic Co-C cleavage

Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247

15

Problems

Methyl donor (Me-H4folate) is too electron

rich to undergo Nu attack

protonation

HN

N NH

N

CH3

R

O

H2N

H

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Problems

Methyl donor (Me-H4folate) is too electron

rich to undergo Nu attack

protonation

Methyl acceptor (thiol of homocysteine, a Nu)

needs to be deprotonated, pKa=10

Zinc help

EXAFS evidence of Zn(II)S3add homocysteine

Zn(II)S4

17

Modified mechanism

Co

CH3

ZnII(S)3HSR

- H+

Co

CH3

ZnII(S)3

S R

Co

ZnII(S)3

H3C S R

H+

Me-H4folateN

HN

HH3C

NH

HN

Co Co Co

CH3

18

Acetogenesis Wood-Ljungdahl Pathway

ACS: acetyl-CoA synthase

CFeSP : corrinoid iron-sulfur protein

contains 4Fe-4S cluster

SNH

NH

OP

O

O O

OH O OH

OP

O

HO O

O

O OH

N

N

N

N

NH2

PHO

HO

O

Acetyl-CoA

O-Demethylase: couples the demethylationof an aromatic methyl ether to

the formation of CH3-HfolateCODH : CO dehydrogenase

Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247

19

Methanogenesis

Reverse of acetyl-CoA synthesis

O

SCoACH4

Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247

20

Reductive Dehalogenase

R Cl 2e- 2H+ RH HCl

Banerjee, R.;

Ragsdale, S. W.

Annu. Rev. Biochem.

2003, 72, 209–247

21

Path A -organocobalt complex formation

or path B -just an electron donor?

No intermediate has been found

Low potential reductants drive the reaction

CoI is the active species

[CoI] disappear as [CoII] appearpath B

kcat(H2O) = 2.3* kcat(D2O)

Cl

HO

e-

Cl

HO

R RH+ Cl

HO

e-

Cl

HO

RR H

HO

RH H

Cl-

Birch reduction

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Isotope

kcat(H2O) /kcat(D2O) = 2.3

Proton inventory is linear

D from solvent is incorporated in the organics

Single proton is

transferred in a

partially rate limiting

reaction

deprotonation

of an active-site

water

Krasotkina, J.; Walters, T.; Maruya, K. A.; Ragsdale, S. W. J. Biol. Chem.

2001, 276, 40991

Isotope inventory plot for the dehalogenation

of 3-chloro-4-hydroxybenzoate