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- W1995533493 abstract "The previously studied complete methyl transfer sequence of tetrahydrofolate-dependent O-demethylation catalyzed by Holophaga foetida strain TMBS4 extracts was separated into two steps using cobalamins as non-physiological substrates: electrochemically produced cob(I) alamin served as methyl acceptor for phenyl methyl ether demethylation, yielding methylcob(III)alamin (reaction I), and methylcob(III)alamin served as donor for tetrahydrofolate methylation, yielding 5-methyl tetrahydrofolate (reaction II). Both reactions were measured with a new and direct photometric assay of cob(I)alamin methylation (or the reverse reaction) at 540 nm, the isobestic wavelength of the cob(II)alamin/cob(I)alamin redox couple (delta epsilon 540 = 4.40 nM-1.cm-1. The rates of reactions I and II were proportional to protein concentration, unlike the complete reaction sequence. Small components of cell extract did not affect activity of reactions I and II. Isovanillate demethylation by extracts of synringate-grown cells (reaction I) required reductive activation by cob(I)alamin and was inhibited and inactivated by cob(II)alamin, indicating that the reaction mechanism was a nucleophilic attack of an enzyme-bound corrinoid in the reduced Co(I) state on the methyl carbon of the ether, rather than a radical attack. Only phenyl methyl ethers were demethylated; demethylation rates were enhanced by ortho-hydroxyl or para-carboxyl groups, but reduced by additional meta substituents. The rate of isovanillate demethylation was 81 nmol.min-1.(mg protein)-1 [0.76 mM cob(I)alamin] and apparent kinetic constants for cob(I)alamin were: Km = 1.2 mM, Vmax = 220 nmol min-1.(mg protein)-1, and Vmax/Km = 180 nmol.min-1.(mg protein) 1.mM-1 3,5-Dihydroxyanisole demethylation by extracts of 3,5-dihydroxyanisole-grown cells (also reaction I) was much slower. Reaction II did not require activation; specific activity and the specificity constant for methylcob(III)alamin were much lower." @default.
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- W1995533493 date "1994-12-01" @default.
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- W1995533493 title "O -Demethylation by the Homoacetogenic Anaerobe Holophaga Foetida Studied by a New Photometric Methylation Assay Using Electrochemically Produced Cob(I)Alamin" @default.
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- W1995533493 doi "https://doi.org/10.1111/j.1432-1033.1994.00945.x" @default.
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