The Reaction Mechanism of Methyl-Coenzyme M Reductase: How an Enzyme Enforces Strict Binding Order [Microbiology]

February 17th, 2015 by Wongnate, T., Ragsdale, S. W.

Methyl-Coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (Coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-sulfanylethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 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:methyl-SCoM) is productive while the other (MCR:CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR:CoB7SH complex is highly disfavored (Kd = 56 mM) However, binding of CoB7SH to the productive MCR:methyl-SCoM complex to form the active ternary complex (CoB7SH:MCR(NiI):CH3SCoM) is highly favored (Kd = 79 μM). Only then can the chemical reaction occur (kobs = 20 s-1 at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(NiII):CoB7S-⋅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.