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 tiss ue damage in neurological, inflammatory, and autoimmune disorders, design o f NOS inhibitors has received much attention. Most inhibitors described to date include a guanidine-like structural motif and interact with the guanid inium region of the L-argnine-binding site. We report here studies with L-a rginine analogs having one or both terminal guanidinium nitrogens replaced by functionalities that preserve some, but not all, of the molecular intera ctions possible for the -NH2, =NH, or =NH2+ groups of L-arginine, Replaceme nt groups include -NH-alkyl, -alkyl, =O, and =S, Binding of L-canavanine, a n analog unable to form hydrogen bonds involving a N-5-proton, was also exa mined. From our results and previous work, we infer the orientation of thes e compounds in the L-arginine-binding site and use IC50 or K-i values and o ptical difference spectra to quantitate their affinity relative to L-argini ne, 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 hydroge n bonding properties of the group bound. The individual H-bonds involved ar e, however, weaker than expected (<2 versus 3-6 kcal), These findings eluci date substrate binding forces in the NOS active site and identify an import ant constraint on NOS inhibitor design.