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 (.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.
Highlights
In mammalian cells only two enzymes are known to require cobalamin 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 syn
When Baby hamster kidney (BHK) cells were grown in Dulbecco’s modified Eagle (DME) supplemented with 10% fetal bovine serum (FBS), but lacking added oxide species. AdoCbl and hydroxycobalamin (OH-Cbl), methylmalonyl-CoA mutase activity in cell extracts was 0.52 Ϯ 0.21 nmol/min/mg of protein (Fig. 1, open bar on left)
When cells were incubated for 16 h in DME medium containing 10% FBS supplemented with 1 M OH-Cbl, enzyme activity in the cell extracts increased to the same level as observed when extracts were incubated with AdoCbl (Fig. 1, open bar on right), and adding AdoCbl to the extract had only a marginal effect (Fig. 1, diagonally striped bar on right)
Summary
Arginine-deficient Eagle’s minimal essential media (MEM) was either from ICN Pharmaceuticals or was made as described previously [22]. High performance liquid chromatography (HPLC) was performed as previously described [23], with C18 reversed-phase columns from Whatman. [1-14C]Propionic acid (53 mCi/mmol) and [1,4-14C]succinic acid (54 mCi/mmol) were from Moravek Biochemicals, and [methyl14C]methylmalonic acid (50 mCi/mmol) and L-[4,5-3H]leucine (40 Ci/ mmol) were from American Radiolabeled Chemicals. DEA-NONOate (2-(N,N-diethylamino)-diazenolate-2-oxide), DETA-NONOate ((Z)-1-[2(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), and L-NAME (NG-nitro-L-arginine methyl ester) were from Alexis Biochemicals. Hemoglobin was made from freshly lysed red blood cells [24]. NO gas, Ͼ99% pure from Matheson Gas Products, was passed through 5 M NaOH immediately prior to use to remove contaminating nitrogen oxide species. AdoCbl and hydroxycobalamin (OH-Cbl) were from Sigma with the AdoCbl protected from light except as indicated
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