INHIBITION OF BOVINE BRAIN NITRIC-OXIDE SYNTHASE BY ALPHA-AMINO AND ALPHA-CARBOXYL DERIVATIVES OF N-G-ALLYL-L-ARGININE

Three derivatives of the mechanism-based inhibitor N-G-allyl-L-arginin e, designed to eliminate the effect of charge on the alpha-functional groups, were synthesized and tested as inhibitors of purified bovine b rain nitric oxide synthase. The inhibitory properties of N-G-allyl-L-a rginine, N-G-allyl-L-arginine methyl ester, N-G-acetyl-N-G-allyl-L-arg inine, and N-alpha-acetyl-N-G-allyl-L-arginine methyl ester were deter mined in steady-state kinetic assays. The K(i)s of the four compounds were 7 +/- 1, 11 +/- 1, 147 +/- 13, and 480 +/- 45 mu M, respectively. These results demonstrate that conversion of the alpha-carboxylgroup of N-G-allyl-L-arginine to a methyl ester had only a small effect on i ts inhibitory properties, whereas acetylation of the alpha-amino group increased the K-i by more than an order of magnitude. Modification of both the alpha-amino and alpha-carboxyl groups increased the K-i more dramatically from 7 to 480 mu M. Derivatization of the alpha-amino an d alpha-carboxyl groups of N-G-allyl-L-arginine would not be expected to alter the chemistry of inactivation by the N-G-allyl guanidine moie ty, and therefore the increased K(i)s 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 alpha-amino group of arginine for binding, but that it can accommodate a modified alpha-carboxylate. Thus, conservative modifica tion at the alpha-carboxyl may represent a starting point for the desi gn and synthesis of other inhibitors targeted at nitric oxide synthase . (C) 1995 Academic Press, Inc.