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-arginine-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 -NH(2), =NH, or =NH(2)(+) 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 IC(50) 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.
Babu
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BR
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Frey
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Griffith
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D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D019791:Guanidine; D006859:Hydrogen; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats
D006859:Hydrogen; D001120:Arginine; D019001:Nitric Oxide Synthase; D019791:Guanidine