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Spectroscopic study of the cobalamin-dependent methionine synthase in the activation conformation: effects of the Y1139 residue and S-adenosylmethionine on the B12 cofactor.

Authors
  • Liptak, Matthew D
  • Datta, Supratim
  • Matthews, Rowena G
  • Brunold, Thomas C
Type
Published Article
Journal
Journal of the American Chemical Society
Publisher
American Chemical Society
Publication Date
Dec 03, 2008
Volume
130
Issue
48
Pages
16374–16381
Identifiers
DOI: 10.1021/ja8038129
PMID: 19006389
Source
Medline
License
Unknown

Abstract

The cobalamin-dependent methionine synthase (MetH) from Escherichia coli is a modular enzyme that catalyzes a methyl group transfer from methyltetrahydrofolate to homocysteine via a methylcob(III)alamin (MeCbl) intermediate, generating tetrahydrofolate and methionine (Met). Once every approximately 2000 turnovers, the cobalamin cofactor is converted to the inactive cob(II)alamin (Co(2+)Cbl) form, from which MeCbl has to be recovered for MetH to re-enter the catalytic cycle. A particularly puzzling aspect of this reactivation process is that it requires the reduction of the Co(2+)Cbl species to cob(I)alamin (Co(1+)Cbl) by flavodoxin, a reaction that would appear to be endergonic on the basis of the corresponding reduction potentials. To explore how MetH may overcome this apparent thermodynamic challenge, we have prepared the I690C/G743C variant of a C-terminal fragment of MetH (MetH(CT)) to lock the enzyme into the activation conformation without perturbing any of the residues in the vicinity of the active site. A detailed spectroscopic characterization of this species and the I690C/G743C/Y1139F MetH(CT) triple mutant reveals that the strategy employed by MetH to activate Co(2+)Cbl for Co(2+) --> Co(1+) reduction likely involves (i) an axial ligand switch to generate a five-coordinate species with an axially coordinated water molecule and (ii) a significant lengthening, or perhaps complete rupture, of the Co-OH(2) bond of the cofactor, thereby causing a large stabilization of the Co 3d(z(2))-based "redox-active" molecular orbital. The lengthening of the Co-OH(2) bond is mediated by the Y1139 active-site residue and becomes much more dramatic when the S-adenosylmethionine substrate is present in the enzyme active site. This substrate requirement provides MetH a means to suppress deleterious side reactions involving the transiently formed Co(1+)Cbl "supernucleophile".

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