O-DEMETHYLATION BY THE HOMOACETOGENIC ANAEROBE HOLOPHAGA-FOETIDA STUDIED BY A NEW PHOTOMETRIC METHYLATION ASSAY USING ELECTROCHEMICALLY PRODUCED COB(I)ALAMIN

The previously studied complete methyl transfer sequence of tetrahydro folate-dependent O-demethylation catalyzed by Holophaga foetida strain TMBS4 extracts was separated into two steps using cobalamins as non-p hysiological substrates: electrochemically produced cob(I)alamin serve d as methyl acceptor for phenyl methyl ether demethylation, yielding m ethylcob(III)alamin (reaction I), and methylcob(III)alamin served as d onor for tetrahydrofolate methylation, yielding 5-methyl tetrahydrofol ate (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 isosbestic wavelength of the cob(II)alamin/cob(I)alam in redox couple (Delta epsilon(540) = 4.40 mM(-1) . cm(-1)). The rates of reactions I and II were proportional to protein concentration, unl ike the complete reaction sequence. Small components of cell extract d id not affect activity of reactions I and II. Isovanillate demethylati on by extracts of syringate-grown cells (reaction I) required reductiv e activation by cob(I)alamin and was inhibited and inactivated by cob( II)alamin, indicating that the reaction mechanism was a nucleophilic a ttack of an enzyme-bound corrinoid in the reduced Co(I) state on the m ethyl carbon of the ether, rather than a radical attack. Only phenyl m ethyl ethers were demethylated; demethylation rates were enhanced by o rtho-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 co nstants for cob(I)alamin were: K-m = 1.2 mM, V-max = 220 nmol min(-1) . (mg protein)-1, and V-max/K-m = 180 nmol . min(-1) . (mg protein)(-1 ) . mM(-1) . 3,5-Dihydroxyanisole demethylation by extracts of 3,5-dih ydroxyanisole-grown cells (also reaction I) was much slower. Reaction II did not require activation; specific activity and the specificity c onstant for methylcob(III)alamin were much lower.