L-arginine binding to nitric-oxide synthase - The role of H-bonds to the nonreactive guanidinium nitrogens

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to nitric oxide and L-citrulline. Because overproduction of nitric oxide causes tissue damage in neurological, inflammatory, and autoimmune disorders, design of NOS inhibitors has received much attention. Most inhibitors described to date include a guanidine-like structural motif and interact with the guanidinium region of the L-argnine-binding site. We report here studies with L-arginine analogs having one or both terminal guanidinium nitrogens replaced by functionalities that preserve some, but not all, of the molecular interactions possible for the -NH2, =NH, or =NH2+ groups of L-arginine, Replacement groups include -NH-alkyl, -alkyl, =O, and =S, Binding of L-canavanine, an analog unable to form hydrogen bonds involving a N-5-proton, was also examined. From our results and previous work, we infer the orientation of these compounds in the L-arginine-binding site and use IC50 or K-i values and optical difference spectra to quantitate their affinity relative to L-arginine, We find that the non-reactive guanidinium nitrogen of L-arginine binds in a pocket that is relatively intolerant of changes in the size or hydrogen bonding properties of the group bound. The individual H-bonds involved are, however, weaker than expected (<2 versus 3-6 kcal), These findings elucidate substrate binding forces in the NOS active site and identify an important constraint on NOS inhibitor design.