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-mercaptoethanesulfonate (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 whereas 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. In conclusion, 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.
Wongnate, Thanyaporn and Ragsdale, Stephen W.. "The reaction mechanism of methyl-coenzyme M reductase: How an enzyme enforces strict binding order." Journal of Biological Chemistry, vol. 290, no. 15, Feb. 2015. https://doi.org/10.1074/jbc.m115.636761
Wongnate, Thanyaporn, & Ragsdale, Stephen W. (2015). The reaction mechanism of methyl-coenzyme M reductase: How an enzyme enforces strict binding order. Journal of Biological Chemistry, 290(15). https://doi.org/10.1074/jbc.m115.636761
Wongnate, Thanyaporn, and Ragsdale, Stephen W., "The reaction mechanism of methyl-coenzyme M reductase: How an enzyme enforces strict binding order," Journal of Biological Chemistry 290, no. 15 (2015), https://doi.org/10.1074/jbc.m115.636761
@article{osti_1348414,
author = {Wongnate, Thanyaporn and Ragsdale, Stephen W.},
title = {The reaction mechanism of methyl-coenzyme M reductase: How an enzyme enforces strict binding order},
annote = {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-mercaptoethanesulfonate (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 whereas 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. In conclusion, 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.},
doi = {10.1074/jbc.m115.636761},
url = {https://www.osti.gov/biblio/1348414},
journal = {Journal of Biological Chemistry},
issn = {ISSN 0021-9258},
number = {15},
volume = {290},
place = {United States},
publisher = {American Society for Biochemistry and Molecular Biology},
year = {2015},
month = {02}}