Substrate Analogues as Mechanistic Probes of Methyl-S-coenzyme M Reductase

Lawrence P. Wackett, John F. Honek, Tadhg P. Begley, William H. Orme-Johnson, Christopher T. Walsh, Valerie Wallace

Research output: Contribution to journalArticlepeer-review

32 Scopus citations


Methyl-S-coenzyme M reductase catalyzes the ultimate methane-yielding reaction in methanogenic bacteria, the reductive cleavage of the terminal carbon-sulfur bond of 2-(methylthio)ethanesulfonic acid. This protein has previously been shown to contain 2 equiv of a tightly bound nickel corphinoid cofactor, denoted cofactor F430, that may play a role in catalysis. Prior to this study, only one substrate analogue, ethyl-S-coenzyme M, had been demonstrated to be processed to a product by anaerobic cell extracts from Methanobacterium thermoautotrophicum strain ΔH. In this investigation, we have synthesized three additional substrate analogues that serve as substrates as well as five previously unknown inhibitors. Steady-state kinetic techniques were developed in order to assess relative rates of processing for these substrates and inhibitors by use of anaerobic cell extracts from M. thermoautotrophicum. With this assay system, a KM of 0.1 mM and a Kcatof 17 min-1 were determined for methyl-S-coenzyme M as substrate. Methyl-seleno-coenzyme M was converted to methane with a kcat threefold higher than that of methyl-S-coenzyme M, but Kcat/KMwasunchanged. The carbon-oxygen bond of 2-methoxyethanesulfonic acid was not cleaved to yield methane, but this analogue acted as an inhibitor with a K1of 8.3 mM. Methyl reductase catalyzed reductive cleavage of difluoromethyl-S-coenzyme M to yield difluoromethane as the sole product, but trifluoromethyl-S-coenzyme M and trifluoromethyl-seleno-coenzyme M were inhibitors and not substrates. Allyl-S-coenzyme M, cyano-S-coenzyme M, and (difluoromethyl sulfoxide)-coenzyme M were shown to be tight binding inhibitors, but no spectroscopically detectable intermediates were observed upon inCubation of these analogues with methyl reductase.

Original languageEnglish (US)
Pages (from-to)6012-6018
Number of pages7
Issue number19
StatePublished - 1987


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