Mechanistic studies of methane biogenesis by methyl-coenzyme M reductase: evidence that coenzyme B participates in cleaving the C-S bond of methyl-coenzyme M.
Methyl-coenzyme M reductase (MCR), the key enzyme in methanogenesis, catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). Steady-state and presteady-state kinetics have been used to test two mechanistic models that contrast in the role of CoBSH in the MCR-catalyzed reaction. In class 1 mechanisms, CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage. On the other hand, in class 2 mechanisms, methane is formed in the absence of CoBSH, which functions to regenerate active MCR after methane is released. Steady-state kinetic studies are most consistent with a ternary complex mechanism in which CoBSH binds before methane is formed, as found earlier [Bonacker et al. (1993) Eur. J. Biochem. 217, 587-595]. Presteady-state kinetic experiments at high MCR concentrations are complicated by the presence of tightly bound CoBSH in the purified enzyme. Chemical quench studies in which (14)CH(3)-SCoM is rapidly reacted with active MCRred1 in the presence versus the absence of added CoBSH indicate that CoBSH is required for a single-turnover of methyl-SCoM to methane. Similar single turnover studies using a CoBSH analogue leads to the same conclusion. The results are consistent with class 1 mechanisms in which CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage and are inconsistent with class 2 mechanisms in which CoBSH binds after methane is formed. These are the first reported pre-steady-state kinetic studies of MCR.
Horng
Y C
YC
Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Becker
D F
DF
Ragsdale
S W
SW
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008697:Methane / Q000737:chemistry* / Q000378:metabolism*; D017014:Methanobacterium / Q000201:enzymology*; D008956:Models, Chemical; D010769:Phosphothreonine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014466:Ultraviolet Rays
C049349:7-mercaptoheptanoylthreonine phosphate; D010769:Phosphothreonine; D008697:Methane