title: The reaction mechanism of methyl-coenzyme M reductase: how an enzyme enforces strict binding order.
abstract: 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(Ni(I))·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(Ni(II))·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.
authors.affiliation: From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
authors.forename: Thanyaporn
authors.initials: T
authors.lastname: Wongnate
authors.affiliation: From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109 sragsdal@umich.edu.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Ragsdale
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D015080:Mesna / Q000737:chemistry / Q000378:metabolism*; D008697:Methane / Q000737:chemistry / Q000378:metabolism; D017013:Methanobacteriaceae / Q000201:enzymology* / Q000235:genetics; D008954:Models, Biological; D008956:Models, Chemical; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010769:Phosphothreonine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D019843:Archaeal Proteins; D021122:Protein Subunits; C049349:7-mercaptoheptanoylthreonine phosphate; D010769:Phosphothreonine; D009532:Nickel; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase; D015080:Mesna; D008697:Methane
keywords: Electron Paramagnetic Resonance (EPR); Enzyme Inhibitor; Enzyme Kinetics; Enzyme Mechanism; Metalloenzyme; Nickel; Pre-steady-state Kinetics