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

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Three derivatives of the mechanism-based inhibitor N G-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 N G-allyl-L-arginine, N G-allyl-L-arginine methyl ester, N α-acetyl- N G-allyl-L-arginine, and N α-acetyl- N G-allyl-L-arginine methyl ester were determined in steady-state kinetic assays. The K i s 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 N G-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 K i by more than an order of magnitude. Modification of both the α-amino and α-carboxyl groups increased the K i more dramatically from 7 to 480 μM. Derivatization of the α-amino and α-carboxyl groups of N G-allyl-L-arginine would not be expected to alter the chemistry of inactivation by the N G-allyl guanidine moiety, 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 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.