NG-Methyl-L-arginine functions as an alternate substrate and mechanism-based inhibitor of nitric oxide synthase

Abstract

NG-Methyl-L-arginine (L-NMA) is one of the most commonly used inhibitors of the nitric oxide synthases (NOS). Results reported here demonstrate that L-NMA is an alternate substrate and a mechanism-based inhibitor of the inducible NOS purified from murine macrophages. The irreversible inhibition displays pseudo-first-order kinetics of inactivation with kinact = 0.07 min-1 and KI = 2.7 microM. Inactivation of NOS is enantiospecific for L-NMA, and substrate protection against inactivation is enantiospecific for L-arginine. L-NMA is hydroxylated, producing NG-hydroxy-NG-methyl-L-arginine (L-NHMA), and both compounds are slow, partially uncoupled alternate substrates for NOS. Processing of L-NMA by NOS results in four amino acid products: L-NHMA, NG-hydroxy-L-arginine (L-NHA), L-arginine, and citrulline. Deformylation of L-NMA and L-NHMA precedes the formation of citrulline and nitric oxide (.NO). Partial uncoupling of NADPH oxidation during L-NMA and L-NHMA processing results in hydrogen peroxide formation. The apparent Km values for L-NMA and L-NHMA are 3.1 and 7.4 microM, respectively. Turnover of L-NMA and L-NHMA to .NO and citrulline is slow relative to L-arginine: Vmax(L-arginine/L-NMA) = 20:1; Vmax(L-arginine)/(L-NHMA) = 13:1. NOS contains a functional cytochrome P-450-type heme, and the formation of these products from L-NMA is consistent with cytochrome P-450 monooxygenase chemistry. Other than the NOS reaction intermediate L-NHA, L-NMA and L-NHMA are the first NG-substituted L-arginines identified as substrates for NOS.