Vitamin B12 Mechanism
Wan-Chun Chung 4/18/2011
Outline


Introduction
Isomerase
mechanism
problems

Methyltranferase
mechanism
problems

Dehalogenase
mechanism
2
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
3
H2NOC
CH3
H2NOC
N R
H3C
H 3C
H
N
Co
N
N
H2NOC
Structure
CONH2
CH3
Corrin ring
CONH2
CH3
CH3
R= deoxyadenosyl
CN-, OH-, H2O
HO
CH3 CH
3
O
C
H2
CONH2
NH
N
CH3
CH3HO
O
O
P
N
O
N
N
N
NH2
Dimethylbenzimidazole
H
O
OH
N
CH3
Base-on/base-off
nucleotide
O
OH
cobalamin
4
Three main types

Isomerase
X H
C C Z
H X
C C Z
Diol dehydrase, ammonia lyase, glutamate mutase,
methylmalonyl CoA mutase, ribonucleotide reductase

Methyltransferase
R CH3
R' H
R H
R' CH3
Methionine synthase, acetyl-CoA synthase

Reductive dehalogenase
R Cl
2e-
2H+
RH
HCl
anaerobic dehalogenase
Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247
5
Isomerase
X H
C C Z
H X
C C Z
Class I : Base-off/His-on
Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247
6
Homolysis v.s. Heterolysis
III
Co
Co
III
CH2Ado
CH2Ado
CoII
CH2Ado
CoI
CH2Ado
RH
RH
CoII
R
CH3Ado
CoI
R
CH3Ado
C
Co
C
C
Co
Co
B
B
B
CoII
d7, l.s.
17 e-
CoI
d8, l.s.
16 e-
CoIII
d6, l.s.
18 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
 Homolytic pathway !
Jordan, R. B. Reaction Mechanisms of Inorganic and Organometallic Systems;
Oxford, 2007
7





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 shortlived
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
8
Isomerase
Class II : Base-on
Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247
9
Problems

Thermal stability of Co-C bond compare to
high reactivity of enzymic reaction
1.
O
H
N
O
N
Co
N
O
N
H
O
(DH)2Co (or
(DMGH)2Co)
2. L(DMGH)2Co-CH3 stretching frequency, L
affects the stability of CoII
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
10
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
11
Problems

Limited precedent for radical rearrangement
Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247
12
Methyltransferase

Methionine synthase
O
CH3
N
R
HN
H3N H
HS
H2N
N
N
H
CH3-H4folate
O
HN
H2N
N
O
O
HN
H
N
H3N H
R
H3CS
O-
H2N
homocysteine
N
H4folate
N
H
O-
methionine
S
CH3
N
R
O
CH3
CoI
N
H
-
NH3
H
O
O
H+
O
HN
H2N
N
H
N
N
H
R
CH3
CoIII
-
S
B
-
NH3
H
O
O
Shriver textbook
13
Nucleophile attack vs Oxidative addition


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
Oxidative addition needs two cis vacant site  impossible
Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247
14
Problems

Methyl donor (Me-H4folate) is too electron
rich to undergo Nu attack
 protonation
HN
H2N
O H CH3
N
R
N
N
H
15
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)S3add homocysteine
Zn(II)S4
16
Modified mechanism
H
N
H+
Me-H4folate
N
H
N
H
H3 C
Co
Co
ZnII(S)3
ZnII(S)3
S R
CH3
Co
HSR
- H+
N
H
CH3
Co
ZnII(S)3
H3C S R
CH3
Co
Co
17
Acetogenesis Wood-Ljungdahl Pathway
NH2
ACS: acetyl-CoA synthase
O
S
CFeSP : corrinoid iron-sulfur protein
N
H
O
contains 4Fe-4S cluster
O-Demethylase: couples the demethylation
of an aromatic methyl ether to
the formation of CH3-Hfolate
CODH : CO dehydrogenase
HO
O
N
H
O
OH
O
P
O
N
O
P
O OH
O
N
N
N
Acetyl-CoA
HO
O OH
P
HO
O
Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247
18
Methanogenesis

O
CH4
SCoA
Reverse of acetyl-CoA synthesis
Banerjee, R.; Ragsdale, S. W. Annu. Rev. Biochem. 2003, 72, 209–247
19
Reductive Dehalogenase
R Cl
2e-
2H+
RH
HCl
Banerjee, R.;
Ragsdale, S. W.
Annu. Rev. Biochem
2003, 72, 209–247
20
Birch reduction
R
Cl
HO
HO
e-





Cl
R
H+
Cl
H
HO
R
H
Cl
HO
e-
R
H
Cl-
R
HO
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] appearpath B
kcat(H2O) = 2.3* kcat(D2O)
21
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
Isotope inventory plot for the dehalogenation
of 3-chloro-4-hydroxybenzoate
deprotonation
of an active-site
water
Krasotkina, J.; Walters, T.; Maruya, K. A.; Ragsdale, S. W. J. Biol. Chem.
2001, 276, 40991
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