Spatial relationship between L-arginine and heme binding sites of endothelial nitric-oxide synthase.
Binding of L-arginine and imidazole to the endothelial nitric-oxide synthase (eNOS) was characterized by direct heme spectral perturbation. L-Arginine is competitive with imidazole for binding to eNOS. Both equilibrium binding and kinetic binding were measured at 4 and 23 degrees C for these two ligands. Kd (imidazole) is 60 microM and 110 microM, kon (imidazole) is 2.5 x 10(5) M-1 s-1 and 1. 2 x 10(6) M-1 s-1, koff (imidazole) is 11.8 s-1 and 116 s-1 at 4 and 23 degrees C, respectively. Corresponding values for L-arginine are calculated from the data of binding competition with imidazole and computer modeling. Kd (L-arginine) is 0.5 microM and 2.0 microM, kon (L-arginine) is 2 x 10(5) M-1 s-1 and 8 x 10(5) M-1 s-1, koff (L-arginine) is 0.08 s-1 and 1.6 s-1 at 4 and 23 degrees C, respectively. It is suggested that binding of both ligands occurs through the same access channel to the heme site based on their similarly slow association rate constants. A series of potential heme ligands and amino acid analogs of L-arginine were evaluated for their binding and their effect on the heme structure. All ligands besides cyanide tested for binding inhibition are competitive with either L-arginine or imidazole. The space for the distal heme ligand was estimated to be approximately 6.3 x 6.7 A by three groups of rigid planar ligands: imidazole, pyridine, and pyrimidine. Results of the thiazole and amino acid ligand series permitted the conclusion that the guanidine group of L-arginine is critical for its binding affinity and its specific orientation relative to the heme. Such a specific conformation is essential for the oxygenase mechanism of eNOS.
Berka
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V
Division of Hematology, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, Texas 77030, USA.
Chen
P F
PF
Tsai
A L
AL
D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D003486:Cyanides / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*
D003486:Cyanides; D007093:Imidazoles; D006418:Heme; C029899:imidazole; D001120:Arginine; D019001:Nitric Oxide Synthase; D008239:Lysine