Inhibition of Bovine Brain Nitric-Oxide Synthase by α-Amino and α-Carboxyl Derivatives of NG-Allyl-L-Arginine

Three derivatives of the mechanism-based inhibitor NG-allyl-L-arginine, designed to eliminate the effect of charge on the α-functional groups, were synthesized and tested as inhibitors of purified bovine brain nitric oxide synthase. The inhibitory properties of NG-allyl-L-arginine, NG-allyl-L-arginine methyl ester, Nα-acetyl-NG-allyl-L-arginine, and Nα-acetyl-NG-allyl-L-arginine methyl ester were determined in steady-state kinetic assays. The Kis of the four compounds were 7 ± 1, 11 ± 1, 147 ± 13, and 480 ± 45 μM, respectively. These results demonstrate that conversion of the α-carboxylgroup of NG-allyl-L-arginine to a methyl ester had only a small effect on its inhibitory properties, whereas acetylation of the α-amino group increased the Ki by more than an order of magnitude. Modification of both the α-amino and α-carboxyl groups increased the Ki more dramatically from 7 to 480 μM. Derivatization of the α-amino and α-carboxyl groups of NG-allyl-L-arginine would not be expected to alter the chemistry of inactivation by the NG-allyl guanidine moiety, and therefore the increased Kis of the derivatives are probably due solely to changes in binding specificity. These data suggest that the arginine binding pocket of brain nitric oxide synthase prefers the unmodified α-amino group of arginine for binding, but that it can accommodate a modified α-carboxylate. Thus, conservative modification at the alpha-carboxyl may represent a starting point for the design and synthesis of other inhibitors targeted at nitric oxide synthase.