Nitric Oxide Inhibits Mammalian Methylmalonyl-CoA Mutase

Abstract

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 (⅐CH 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 (>600 M) 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 ⅐CH 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 ⅐CH 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.In mammalian cells only two enzymes are known to require cobalamin (vitamin B 12 ) as a cofactor: methionine synthase, which uses methylcobalamin, and methylmalonyl-coenzyme A (CoA) mutase, which uses 5Ј-deoxyadenosyl-cobalamin (AdoCbl).1 During the reaction catalyzed by methionine synthase, methylcobalamin is cleaved heterolytically generating cobalamin I (Cbl(I)) and a methyl carbocation, whereas during the reaction catalyzed by methylmalonyl-CoA mutase, AdoCbl is cleaved homolytically generating cobalamin II (Cbl(II)) and a deoxyadenosyl radical (⅐CH 2 -Ado) (1, 2). The latter serves to abstract a hydrogen atom from the substrate methylmalonylCoA, and after intramolecular migration of a thioester moiety, the hydrogen atom is transferred back to generate the product succinyl-CoA. In the final step of the reaction, the deoxyadenosyl radical recombines with Cbl(II) to regenerate AdoCbl (3). It should be noted that the homolytic cleavage of AdoCbl occurs essentially only on substrate binding to the enzyme (3). Nitric oxide (NO) is produced by a variety of mammalian cell types, and has a diverse array of cellular and physiological functions including regulation of cell growth, differentiation, and apoptosis, and modulation of blood pressure, platelet aggregation, and synaptic plasticity (4 -6). At pharmacological concentrations, NO reacts with several different chemical groups, but at phys...

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Nitroxylcob(III)alamin: Synthesis and X‐ray Structural Characterization

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