The Mechanism of Adenosylmethionine-Dependent Activation of Methionine Synthase: A Rapid Kinetic Analysis of Intermediates in Reductive Methylation of Cob(II)alamin Enzyme

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• 作者：Joseph T. Jarrett ; David M. Hoover ; Martha L. Ludwig ; and Rowena G. Matthews
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：September 8, 1998
• 年：1998
• 卷：37
• 期：36
• 页码：12649 - 12658
• 全文大小：157K
• 年卷期：v.37,no.36(September 8, 1998)
• ISSN：1520-4995

文摘

Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group frommethyltetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. During this primaryturnover cycle, the enzyme alternates between the active methylcobalamin and cob(I)alamin forms of theenzyme. Formation of the cob(II)alamin prosthetic group by oxidation of cob(I)alamin or photolysis ofmethylcobalamin renders the enzyme inactive. Methionine synthase from E. coli catalyzes its ownreactivation by a reductive methylation that involves electron transfer from reduced flavodoxin and methyltransfer from AdoMet. This process has been proposed to involve formation of a transient cob(I)alaminintermediate that is then trapped by methyl transfer from AdoMet. During aerobic growth of E. coli,electrons for this process are ultimately derived from NADPH, and electron transfer does not generate adetectable level of cob(I)alamin due to the large potential difference between the NADPH/NADP⁺ coupleand the cob(I)alamin/cob(II)alamin couple. In this paper, we show that even in the presence of the strongreductant flavodoxin hydroquinone, cob(I)alamin is not observed as a significant intermediate. Wedemonstrate, however, that this is due to a rate-limiting reorganization of the cobalt ligand environmentfrom five-coordinate to four-coordinate cob(II)alamin. Mutation of aspartate 757 to glutamate results ina cob(II)alamin enzyme that is ~70% four-coordinate, and reductive methylation of this enzyme usingflavodoxin hydroquinone as the electron donor proceeds through a kinetically competent cob(I)alaminintermediate. Furthermore, wild-type cob(I)alamin enzyme produced by chemical reduction reacts withAdoMet in a kinetically competent reaction. We provide evidence that methyl transfer from AdoMet tocob(I)alamin enzyme results initially in formation of a five-coordinate methylcobalamin enzyme that slowlydecays to the active six-coordinate methylcobalamin enzyme. We propose a kinetic scheme for reductivemethylation of wild-type cob(II)alamin enzyme by adenosylmethionine and flavodoxin hydroquinone inwhich slow conformational changes mask the relatively fast electron and methyl transfer steps.