The Reaction Mechanism of Methyl-Coenzyme M Reductase HOW AN ENZYME ENFORCES STRICT BINDING ORDER

Methyl-coenzyme M reductase (MCR) is a nickel tetrahydro-corphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH, and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR center dot methyl-SCoM) is productive whereas the other (MCR center dot CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory NICR center dot CoB7SH complex is highly disfavored (K-d = 56 mm). However, binding of CoB7SH to the productive MCR center dot methyl-SCoM complex to form the active ternary complex (CoB7SH.MCR(Ni-1)center dot CH3SCoM) is highly favored (K-d = 79 mu m). Only then can the, chemical reaction occur (k(obs) = 20 s(-1) at 25 degrees C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(Ni-II)center dot CoB7S-center dot SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates.