NATURE OF THE LOW ACTIVITY OF S-METHYL-COENZYME-M REDUCTASE AS DETERMINED BY ACTIVE-SITE TITRATIONS

Purified S-methyl-coenzyme M reductase (methyl-reductase) exhibits a v ery low fraction of its in vivo activity, suggesting either enzyme ina ctivation during cell lysis and chromatographic purification or the la ck of an activating component in assay mixtures. Evidence that all met hylreductase molecules in the purified protein can catalyze slow subst rate turnover is found in a study of turnover-dependent in vitro incor poration of radiolabeled HS-CoM at the enzyme active site (Hartzell, P . L., Donnelly, M. I., and Wolfe, R. S. (1987) J. Biol. Chem. 262, 558 1-5586). We have conducted active site titrations of purified methylre ductase and of a highly active partially purified preparation (Rospert , S., Bocher, R., Albracht, S. P. J., and Thauer, R. K. (1991) FEBS Le tt. 291, 371-375) using the reversible competitive inhibitor bromoprop anesulfonate (K(i) = 0.05 muM). Curve fitting the data based on an equ ilibrium binding model shows that 0.1-1.4% of purified methylreductase has functional inhibitor binding sites while up to 25% of a highly ac tive preparation binds the inhibitor. An EPR titration of highly activ e methylreductase with this inhibitor is consistent with this result, showing that the MCR-red1 and -red2 EPR signals (Albracht, S. P. J., A nkel-Fuchs, D., Bocher, R., Ellermann, J., Moll, J., van der Zwann, J. W., and Thauer, R. K. (1988) Biochim. Biophys. Acta 955, 86-102) are titrated in parallel with this active fraction. Attempts to observe tu rnover-dependent uptake of radiolabel from [thio-S-35]2-methylthioetha ne-sulfonate by methylreductase were unsuccessful. These results sugge st that the low activity of purified methylreductase is due primarily to low percentages of catalytically competent enzyme.