Dissecting the Catalytic Mechanism of Betaine-Homocysteine S-Methyltransferase by Use of Intrinsic Tryptophan Fluorescence and Site-Directed Mutagenesis
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- 作者：Carmen Castro ; Alejandra A. Gratson ; John C. Evans ; Jiri Jiracek ; Michaela Collinsová ; Martha L. Ludwig ; and Timothy A. Garrow
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：May 11, 2004
- 年：2004
- 卷：43
- 期：18
- 页码：5341 - 5351
- 全文大小：361K
- 年卷期：v.43,no.18(May 11, 2004)
- ISSN：1520-4995
文摘
Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzesthe transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine(DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceedsthrough the formation of a ternary complex, with a transition state mimicked by the inhibitorS-(
-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence todetermine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that theenzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcyis the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to theenzyme to form binary complexes with Kd values of 7.9, 6.9, and 0.28 
M, respectively. Binary complexeswith Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly toBHMT-Hcy to form ternary complexes with Kd values of 1.1 and 0.73 M, respectively. Mutation ofeach of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoestwo distinct conformational changes that are reflected in the fluorescence of the enzyme. The first isinduced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT,the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacingthese residues by site-directed mutagenesis significantly reduces the catalytic efficiency (Vmax/Km) of theenzyme. Replacing Tyr77 with Phe abolishes enzyme activity.
-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence todetermine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that theenzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcyis the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to theenzyme to form binary complexes with Kd values of 7.9, 6.9, and 0.28 M, respectively. Binary complexeswith Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly toBHMT-Hcy to form ternary complexes with Kd values of 1.1 and 0.73 M, respectively. Mutation ofeach of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoestwo distinct conformational changes that are reflected in the fluorescence of the enzyme. The first isinduced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT,the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacingthese residues by site-directed mutagenesis significantly reduces the catalytic efficiency (Vmax/Km) of theenzyme. Replacing Tyr77 with Phe abolishes enzyme activity.