Betaine-homocysteine S-methyltransferase (BHMT) transfers a methyl group from betaine to Hcy to form DMG (dimethylglycine) and Met. The reaction is ordered Bi Bi; Hcy is the first substrate to bind and Met is the last product off. Using intrinsic 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, indicating that the conformational change associated with Hcy binding creates the betaine-binding site. CBHcy [S-(δ-carboxybutyl)-D,L-homocysteine] is a bisubstrate analogue that causes BHMT to adopt the same conformation as the ternary complexes. 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 large peptides, excising a seven-residue peptide within loop L2. CBHcy but not Met binding slows down proteolysis by trypsin. These findings suggest that L2 is involved in the conformational change associated with occupancy at the betaine-binding site and that this conformational change and/or occupancy at both ligand-binding sites protect the enzyme from oxidative inactivation.
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December 2005
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Research Article|
December 06 2005
Conformation-dependent inactivation of human betaine-homocysteine S-methyltransferase by hydrogen peroxide in vitro
Catherine M. Miller;
Catherine M. Miller
1Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, U.S.A.
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Sandra S. Szegedi;
Sandra S. Szegedi
1Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, U.S.A.
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Timothy A. Garrow
Timothy A. Garrow
1
1Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, U.S.A.
1To whom correspondence should be addressed, at 463 Bevier Hall, 905 S. Goodwin Avenue, Urbana, IL 61801, U.S.A. (email [email protected]).
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Publisher: Portland Press Ltd
Received:
February 28 2005
Revision Received:
June 20 2005
Accepted:
July 25 2005
Accepted Manuscript online:
July 25 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 392 (3): 443–448.
Article history
Received:
February 28 2005
Revision Received:
June 20 2005
Accepted:
July 25 2005
Accepted Manuscript online:
July 25 2005
Citation
Catherine M. Miller, Sandra S. Szegedi, Timothy A. Garrow; Conformation-dependent inactivation of human betaine-homocysteine S-methyltransferase by hydrogen peroxide in vitro. Biochem J 15 December 2005; 392 (3): 443–448. doi: https://doi.org/10.1042/BJ20050356
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