NO synthase isozymes have distinct substrate binding sites.
The resonance Raman spectra of the carbon monoxide (CO) derivatives of nitric oxide synthases (NOSs), in which CO coordinates to the heme at the site occupied by oxygen under physiological conditions, are very sensitive to the presence of substrates and inhibitors. Significant differences in the modes associated with the bound CO are now found to depend on the isoenzyme. In the presence of L-arginine, the physiological substrate, the frequencies of the Fe-Co stretching mode and the C-O stretching mode in nNOS, the brain enzyme, are detected at 503 and 1929 cm-1, respectively; whereas in iNOS, the inducible enzyme from macrophage, the modes are detected at 512 and 1906 cm-1, respectively. The frequencies in eNOS, the endothelial isozyme, are similar to those of iNOS. These results indicate that nNOS has a much more open substrate-binding pocket than iNOS and eNOS. A theoretical simulation based on the interaction between the CO and a positively charged guanidino group on the arginine indicates that the polar environment of the CO differs markedly between the isozymes. This may be accounted for either by an arginine-CO distance that is as much as 1 A greater in nNOS than in iNOS and eNOS or by a substantial shielding of the charge on the arginine in nNOS as compared to the other isozymes. This is the first reported detection of a structural difference of the substrate binding sites between the isozymes and serves as an initial step in a rational drug design for NOS.
Fan
B
B
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Wang
J
J
Stuehr
D J
DJ
Rousseau
D L
DL
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002248:Carbon Monoxide / Q000378:metabolism; D002417:Cattle; D003545:Cysteine; D004730:Endothelium, Vascular / Q000201:enzymology; D006418:Heme / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D013059:Spectrum Analysis, Raman
D007527:Isoenzymes; D006418:Heme; D002248:Carbon Monoxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D003545:Cysteine