Nitric oxide inhibits mammalian methylmalonyl-CoA mutase.
Methylmalonyl-CoA mutase is a key enzyme in intermediary metabolism, and children deficient in enzyme activity have severe metabolic acidosis. We found that nitric oxide (NO) inhibits methylmalonyl-CoA mutase activity in rodent cell extracts. The inhibition of enzyme activity occurred within minutes and was not prevented by thiols, suggesting that enzyme inhibition was not occurring via NO reaction with cysteine residues to form nitrosothiol groups. Enzyme inhibition was dependent on the presence of substrate, implying that NO was reacting with cobalamin(II) (Cbl(II)) and/or the deoxyadenosyl radical (.CH(2)-Ado), both of which are generated from the co-factor of the enzyme, 5'-deoxyadenosyl-cobalamin (AdoCbl), on substrate binding. Consistent with this hypothesis was the finding that high micromolar concentrations (> or =600 microm) of oxygen also inhibited enzyme activity. To study the mechanism of NO reaction with AdoCbl, we simulated the enzymatic reaction by photolyzing AdoCbl, and found that even at low NO concentrations, NO reacted with both the generated Cbl(II) and .CH(2)-Ado indicating that NO could effectively compete with the back formation of AdoCbl. Thus, NO inhibition of methylmalonyl-CoA mutase appeared to be from the reaction of NO with both AdoCbl intermediates (Cbl(II) and .CH(2)-Ado) generated during the enzymatic reaction. The inhibition of methylmalonyl-CoA mutase by NO was likely of physiological relevance because a NO donor inhibited enzyme activity in intact cells, and scavenging NO from cells or inhibiting cellular NO synthesis increased methylmalonyl-CoA mutase activity when measured subsequently in cell extracts.
Kambo
Amanpreet
A
Department of Medicine, and Cancer Center, University of California, 9500 Gilman Dr., La Jolla, California 92093-0652, USA.
Sharma
Vijay S
VS
Casteel
Darren E
DE
Woods
Virgil L
VL
Pilz
Renate B
RB
Boss
Gerry R
GR
D000818:Animals; D001120:Arginine / Q000737:chemistry; D002460:Cell Line; D049109:Cell Proliferation; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D008623:Mercaptoethanol / Q000494:pharmacology; D008765:Methylmalonyl-CoA Mutase / Q000037:antagonists & inhibitors*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D020030:Nitric Oxide Donors; D009584:Nitrogen / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011422:Propionates / Q000737:chemistry; D011485:Protein Binding; D013053:Spectrophotometry; D019802:Succinic Acid / Q000737:chemistry; D013997:Time Factors
D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D011422:Propionates; D009569:Nitric Oxide; D008623:Mercaptoethanol; D001120:Arginine; D019802:Succinic Acid; D008765:Methylmalonyl-CoA Mutase; C029658:propionic acid; D009584:Nitrogen; D010100:Oxygen; D019331:NG-Nitroarginine Methyl Ester