ACTIVE-SITE STRUCTURE-ANALYSIS OF RECOMBINANT HUMAN INDUCIBLE NITRIC-OXIDE SYNTHASE USING IMIDAZOLE

Nitric oxide synthase catalyzes the pyridine nucleotide-dependent oxid ation of L-arginine to nitric oxide and L-citrulline. It is a speciali zed cytochrome P-450 monooxygenase that is sensitive to inhibition by imidazole. Steady-state kinetic studies on recombinant human inducible nitric oxide synthase (rH-iNOS) demonstrate that imidazole and l-phen ylimidazole are competitive and reversible inhibitors versus L-arginin e. Structure-activity relationship and pH dependence studies on the in hibition suggest that the neutral form of imidazole may be the preferr ed species and that the only modifications allowed without the loss of inhibition are at the N-1 position of imidazole. Optical spectrophoto metric studies of rH-iNOS with imidazole and 1-phenylimidazole yielded type II difference spectra exhibiting K-d values of 63 +/- 2 and 28 /- 3 mu M, respectively. These values were in good agreement with the steady-state K-i of 95 +/- 10 and 38 +/- 4 mu M, respectively, and con firms the site of binding is at the sixth axial ligand of the heme. Im idazole (2.2 mM) also perturbed the K-d of L-arginine from 3.03 +/- 0. 45 to 209 +/- 10 mu M The observed increase in the K-d for L-arginine is consistent with imidazole being a competitive inhibitor versus L-ar ginine. The IC50 values of imidazole and 1-phenylimidazole were lower in the absence of exogenous BH4, and both inhibitors also competitivel y inhibited the BH4-dependent activation of the enzyme. These data tak en together suggest that the L-arginine, dioxygen, and the BH4 binding sites are in close proximity in rH-iNOS. Furthermore, these studies d emonstrate the usefulness of imidazole compounds as active site probes for recombinant human iNOS.