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Conformation-dependent inactivation of human betaine-homocysteine S-methyltransferase by hydrogen peroxide in vitro

Authors
Publisher
Portland Press Ltd.
Publication Date
Source
PMC
Keywords
  • Research Article
Disciplines
  • Biology
  • Chemistry

Abstract

bic173.dvi Biochem. J. (2005) 392, 443–448 (Printed in Great Britain) doi:10.1042/BJ20050356 443 Conformation-dependent inactivation of human betaine-homocysteine S-methyltransferase by hydrogen peroxide in vitro Catherine M. MILLER, Sandra S. SZEGEDI and Timothy A. GARROW1 Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, U.S.A. Betaine-homocysteine S-methyltransferase (BHMT) transfers a methyl group from betaine to Hcy to form DMG (dimethyl- glycine) and Met. The reaction is ordered Bi Bi; Hcy is the first substrate to bind and Met is the last product off. Using in- trinsic tryptophan fluorescence [Castro, Gratson, Evans, Jiracek, Collinsova, Ludwig and Garrow (2004) Biochemistry 43, 5341– 5351], it was shown that BHMT exists in three steady-state conformations: enzyme alone, enzyme plus occupancy at the first substrate-binding site (Hcy or Met), or enzyme plus occupancy at both substrate-binding sites (Hcy plus betaine, or Hcy plus DMG). Betaine or DMG alone do not bind to the enzyme, indi- cating that the conformational change associated with Hcy binding creates the betaine-binding site. CBHcy [S-(δ-carboxy- butyl)-D,L-homocysteine] is a bisubstrate analogue that causes BHMT to adopt the same conformation as the ternary com- plexes. We report that BHMT is susceptible to conformation- dependent oxidative inactivation. Two oxidants, MMTS (methyl methanethiosulphonate) and hydrogen peroxide (H2O2), cause a loss of the enzyme’s catalytic Zn (Zn2+ ion) and a correlative loss of activity. Addition of 2-mercaptoethanol and exogenous Zn after MMTS treatment restores activity, but oxidation due to H2O2 is irreversible. CD and glutaraldehyde cross-linking indicate that H2O2 treatment causes small perturbations in secondary structure but no change in quaternary structure. Oxidation is attenuated when both binding sites are occupied by CBHcy, but Met alone has no effect. Partial digestion of ligand-free BHMT with trypsin produces two l

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