Betaine-homocysteine methyl transferase (BHMT) catalyzes the synthesis of methionine from betaine and homocysteine (Hcy), utilizing a zinc ion to activate Hcy. BHMT is a key liver enzyme that is important for homocysteine homeostasis. X-ray structures of human BHMT in its oxidized (Zn-free) and reduced (Zn-replete) forms, the latter in complex with the bisubstrate analog, S(delta-carboxybutyl)-L-homocysteine, were determined at resolutions of 2.15 A and 2.05 A. BHMT is a (beta/alpha)(8) barrel that is distorted to construct the substrate and metal binding sites. The zinc binding sequences G-V/L-N-C and G-G-C-C are at the C termini of strands beta6 and beta8. Oxidation to the Cys217-Cys299 disulfide and expulsion of Zn are accompanied by local rearrangements. The structures identify Hcy binding fingerprints and provide a prototype for the homocysteine S-methyltransferase family.
Dedicated to Professor Duilio Arigoni on the occasion of his 75th birthday.−I find the reaction catalyzed by cobalamin-dependent methionine synthase improbable and that catalyzed by cobalamin-independent methionine synthase impossible.× Duilio Arigoni to Rowena Matthews, Z¸rich, 1989 Two enzymes in Escherichia coli, cobalamin-independent methionine synthase (MetE) and cobalamindependent methionine synthase (MetH), catalyze the conversion of homocysteine (Hcy) to methionine using N(5)-methyltetrahydrofolate (CH 3 -H 4 folate) as the Me donor. Despite the absence of sequence homology, these enzymes employ very similar catalytic strategies. In each case, the pK a for the SH group of Hcy is lowered by coordination to Zn 2 , which increases the concentration of the reactive thiolate at neutral pH. In each case, activation of CH 3 -H 4 folate appears to involve protonation at N(5). CH 3 -H 4 folate remains unprotonated in binary E ¥ CH 3 -H 4 folate complexes, and protonation occurs only in the ternary E ¥ CH 3 -H 4 folate ¥ Hcy complex in MetE, or in the ternary E ¥ CH 3 -H 4 folate ¥ cob(I)alamin complex in MetH. Surprisingly, the similarities are proposed to extend to the structures of these two unrelated enzymes. The structure of a homologue of the Hcybinding region of MetH, betaineÀhomocysteine methyltransferase, has been determined. A search of the threedimensional-structure data base by means of the structure-comparison program DALI indicates similarity of the BHMT structure with that of uroporphyrin decarboxylase (UroD), a homologue of the MT2-A and MT2-M proteins from Archaea, which catalyze Me transfers from methylcorrinoids to coenzyme M and share the Znbinding scaffold of MetE. Here, we present a model for the Zn binding site of MetE, obtained by grafting the Zn ligands of MT2-A onto the structure of UroD.
Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the 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 proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-(delta-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K(d) values of 7.9, 6.9, and 0.28 microM, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K(d) values of 1.1 and 0.73 microM, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced 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. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V(max)/K(m)) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.
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