STUDIES ON METHYL-CHLORIDE DEHALOGENASE AND O-DEMETHYLASE IN CELL-EXTRACTS OF THE HOMOACETOGEN STRAIN MC BASED ON A NEWLY DEVELOPED COUPLEDENZYME ASSAY

An enzyme assay was developed to determine the activities of methyl ch loride dehalogenase and O-demethylase of the homoacetogen strain MC. T he formation of methyl tetrahydrofolate from tetrahydrofolate and meth yl chloride or from tetrahydrofolate and vanillate was coupled to the oxidation of methyl tetrahydrofolate to methylene tetrahydrofolate med iated by methylene tetrahydrofolate reductase purified from Peptostrep rococcus productus (strain Marburg) and to the subsequent oxidation of methylene tetrahydrofolate to methenyl tetrahydrofolate catalyzed by methylene tetrahydrofolate dehydrogenase purified from the same organi sm. To drive the endergonic methyl tetrahydrofolate oxidation with NAD (+) as an electron acceptor, the NADH formed in this reaction was reox idized in the exergonic lactate dehydrogenase reaction. The formation of NADPH and methenyl tetrahydrofolate in the methylene tetrahydrofola te dehydrogenase reaction was followed photometrically at 350 nm; epsi lon(350) was about 29.5 mM(-1)cm(-1) (pH 6.5). Using the coupled enzym e assay, the cofactor requirements, the apparent kinetic parameters, t he pH and temperature optima of both enzymes, and the effect of inhibi tors were determined. The activity of methyl chloride dehalogenase and of O-demethylase was dependent on the presence of ATP; arsenate sever ely inhibited both enzyme activities in the absence of ATP. The couple d enzyme assay described allows purification and characterization of m ethyl chloride dehalogenase and O-demethylase and is also appropriate for the enzymatic determination of methyl tetrahydrofolate.