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 °C for these two ligands. K(d) (imidazole) is 60 μM and 110 μM, k(on) (imidazole) is 2.5 x 105 M-1 s-1 and 1.2 x 106 M-1 s-1, k(off) (imidazole) is 11.8 s-1 and 116 s-1 at 4 and 23 °C, respectively. Corresponding values for L- arginine are calculated from the data of binding competition with imidazole and computer modeling. K(d) (L-arginine) is 0.5 μM and 2.0 μM, k(on) (L- arginine) is 2 x 105 M-1 s-1 and 8 x 105 M-1 s-1, k(off) (L- arginine) is 0.08 s-1 and 1.6 s-1 at 4 and 23 °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 ~6.3 x 6.7 Å 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.
CITATION STYLE
Berka, V., Chen, P. F., & Tsai, A. L. (1996). Spatial relationship between L-arginine and heme binding sites of endothelial nitric-oxide synthase. Journal of Biological Chemistry, 271(52), 33293–33300. https://doi.org/10.1074/jbc.271.52.33293
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