TY - JOUR
T1 - Substrate Analogues as Mechanistic Probes of Methyl-S-coenzyme M Reductase
AU - Wackett, Lawrence P.
AU - Honek, John F.
AU - Begley, Tadhg P.
AU - Orme-Johnson, William H.
AU - Walsh, Christopher T.
AU - Wallace, Valerie
PY - 1987
Y1 - 1987
N2 - 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.
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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U2 - 10.1021/bi00393a010
DO - 10.1021/bi00393a010
M3 - Article
C2 - 3120769
AN - SCOPUS:0023662534
SN - 0006-2960
VL - 26
SP - 6012
EP - 6018
JO - Biochemistry
JF - Biochemistry
IS - 19
ER -