Nature of the low activity of S-methyl-coenzyme M reductase as determined by active site titrations.
Purified S-methyl-coenzyme M reductase (methylreductase) exhibits a very low fraction of its in vivo activity, suggesting either enzyme inactivation during cell lysis and chromatographic purification or the lack of an activating component in assay mixtures. Evidence that all methylreductase molecules in the purified protein can catalyze slow substrate turnover is found in a study of turnover-dependent in vitro incorporation of radiolabeled HS-CoM at the enzyme active site (Hartzell, P. L., Donnelly, M. I., and Wolfe, R. S. (1987) J. Biol. Chem. 262, 5581-5586). We have conducted active site titrations of purified methylreductase and of a highly active partially purified preparation (Rospert, S., Bocher, R., Albracht, S. P. J., and Thauer, R. K. (1991) FEBS Lett. 291, 371-375) using the reversible competitive inhibitor bromopropanesulfonate (K(i) = 0.05 microM). Curve fitting the data based on an equilibrium binding model shows that 0.1-1.4% of purified methylreductase has functional inhibitor binding sites while up to 25% of a highly active preparation binds the inhibitor. An EPR titration of highly active methylreductase with this inhibitor is consistent with this result, showing that the MCR-red1 and -red2 EPR signals (Albracht, S. P. J., Ankel-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 turnover-dependent uptake of radiolabel from [thio-35S]2-methylthioethane-sulfonate by methylreductase were unsuccessful. These results suggest that the low activity of purified methylreductase is due primarily to low percentages of catalytically competent enzyme.
Department of Chemistry, University of Georgia, Athens 30602.
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D000476:Alkanesulfonates / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019605:Euryarchaeota / Q000201:enzymology; D015080:Mesna / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010769:Phosphothreonine / Q000031:analogs & derivatives / Q000494:pharmacology
D000476:Alkanesulfonates; D004220:Disulfides; C049349:7-mercaptoheptanoylthreonine phosphate; D010769:Phosphothreonine; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase; D015080:Mesna; D004229:Dithiothreitol