Abstract
The objective of this study was to elucidate the mechanism by which nitric oxide (NO) inhibits NO synthase. Previous studies revealed that NO inhibits unpurified preparations of NO synthase. In the present study, the mechanism by which NO inhibits purified neuronal NO synthase from rat cerebellum was examined. The rate of L-citrulline formation from L-arginine was non-linear despite the presence of excess substrate and cofactors and was further inhibited by 30% by 200 units/ml superoxide dismutase. In contrast, 30 microM oxyhemoglobin increased NO synthase activity by 2-fold and made the reaction rate linear. These observations were consistent with the hypothesis that enzymatically generated NO inhibits NO synthase activity. Exogenous NO (0.1-10 microM) (but not NO2, nitrite, or nitrate) also inhibited NO synthase, and enzyme inhibition was not competitive with L-arginine. NO synthase inhibition by NO and other heme ligands supports the view that heme is involved in the catalytic activity of NO synthase. Oxyhemoglobin prevented but could not reverse enzyme inhibition by NO. NO synthase inhibition by NO was markedly diminished and reversed, however, by tetrahydrobiopterin (50 microM) or a tetrahydrobiopterin-regenerating system, and the latter made the reaction rate linear. In contrast, NO synthase inhibition by NO was markedly enhanced by heme oxidants (10 microM methylene blue; 3 microM ferricyanide), and these oxidants directly inhibited NO synthase activity. These observations suggest that NO interacts with enzyme-bound ferric heme to inhibit NO synthase activity. In support of this view, NO inhibited enzyme activity in the absence of turnover, when the heme iron is in the ferric state, and this inhibition was reversed by tetrahydrobiopterin. Therefore, the oxidation state of heme iron appears to be one important determinant for the inhibitory action of NO, and tetrahydrobiopterin may increase NO synthase activity by diminishing the inhibitory action of NO.
Highlights
Enzyme fractions were diluted 4-fold, heated at 95 "C for 5 min in 0.0625 M Tris-HC1, pH 6.8c, ontaining 10%(w/v) glycerol2,%(w/v) sodium dodecyl sulfate (SDS), 5% (v/v) 2-mercaptoethanol, and 0.001% (w/v)bromphenol blue, and rate of L-citrulline formation was non-linear during a 10-min incubation period at 37 "C (Fig. 1)
This was not attributed to substrate/cofactor depletion or enzyme instability as reaction rates remained non-linear after increasing the concentrations of L-arginine,NADPH, FAD, and calmodulin 5-fold applied to the stacking gel
In ordertodeterminewhethersuperoxideanion, which could baeproduct of the enzymatic reactio(n13, 16, 171, was responsible for the non-linear reactiornates, superoxide dismutase was added to reaction mixtures
Summary
The mechanism by which NO inhibits purified neuronal NO synthase from rat cerebellum was examined. The approach that was taken was to study theeffects of exogenous NO, as well as enzymatically generated NO on purified preparationsof constitutive NO syncould not reverse enzyme inhibition by NO. M~ ethylene blue; 3 JIM ferricyanide), and these oxidants directly inhibited NO synthase activity. These observations suggest that NO interacts with enzyme-boundferric heme to inhibit NO synthase activity. In support of this view, NO inhibited enzyme activity in marked inhibitory effect on purified preparations of NO synthase. The absence of turnover, when the heme iron is in the ferric statea, nd this inhibition was reversed by tetrahy-
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