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

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Abstract

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
JournalBiochemistry
Volume26
Issue number19
DOIs
StatePublished - Jan 1 1987

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Mesna
Oxidoreductases
Substrates
Methane
sulfoxide
Cell Extracts
Carbon
Methanobacterium
methyl coenzyme M reductase
Nickel
Catalysis
Sulfur
Methanogens
Oxygen
Bacteria
Acids
methyl coenzyme M
Assays

Cite this

Wackett, L. P., Honek, J. F., Begley, T. P., Orme-Johnson, W. H., Walsh, C. T., & Wallace, V. (1987). Substrate Analogues as Mechanistic Probes of Methyl-S-coenzyme M Reductase. Biochemistry, 26(19), 6012-6018. https://doi.org/10.1021/bi00393a010

Substrate Analogues as Mechanistic Probes of Methyl-S-coenzyme M Reductase. / Wackett, Lawrence P.; Honek, John F.; Begley, Tadhg P.; Orme-Johnson, William H.; Walsh, Christopher T.; Wallace, Valerie.

In: Biochemistry, Vol. 26, No. 19, 01.01.1987, p. 6012-6018.

Research output: Contribution to journalArticle

Wackett, LP, Honek, JF, Begley, TP, Orme-Johnson, WH, Walsh, CT & Wallace, V 1987, 'Substrate Analogues as Mechanistic Probes of Methyl-S-coenzyme M Reductase', Biochemistry, vol. 26, no. 19, pp. 6012-6018. https://doi.org/10.1021/bi00393a010
Wackett LP, Honek JF, Begley TP, Orme-Johnson WH, Walsh CT, Wallace V. Substrate Analogues as Mechanistic Probes of Methyl-S-coenzyme M Reductase. Biochemistry. 1987 Jan 1;26(19):6012-6018. https://doi.org/10.1021/bi00393a010
Wackett, Lawrence P. ; Honek, John F. ; Begley, Tadhg P. ; Orme-Johnson, William H. ; Walsh, Christopher T. ; Wallace, Valerie. / Substrate Analogues as Mechanistic Probes of Methyl-S-coenzyme M Reductase. In: Biochemistry. 1987 ; Vol. 26, No. 19. pp. 6012-6018.
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abstract = "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.",
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AB - 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.

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