Active-Site Structure Analysis of Recombinant Human Inducible Nitric Oxide Synthase Using Imidazole

Abstract

Nitric oxide synthase catalyzes the pyridine nucleotide-dependent oxidation of L-arginine to nitric oxide and L-citrulline. It is a specialized cytochrome P450 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 1-phenylimidazole are competitive and reversible inhibitors versus L-arginine. Structure-activity relationship and pH dependence studies on the inhibition suggest that the neutral form of imidazole may be the preferred species and that the only modifications allowed without the loss of inhibition are at the N-1 position of imidazole. Optical spectrophotometric studies of rH-iNOS with imidazole and 1-phenylimidazole yielded type II difference spectra exhibiting Kd values of 63 +/- 2 and 28 +/- 3 microM, respectively. These values were in good agreement with the steady-state Ki of 95 +/- 10 and 38 +/- 4 microM, respectively, and confirms the site of binding is at the sixth axial ligand of the heme. Imidazole (2.2 mM) also perturbed the Kd of L-arginine from 3.03 +/- 0.45 to 209 +/- 10 microM. The observed increase in the Kd for L-arginine is consistent with imidazole being a competitive inhibitor versus L-arginine. The IC50 values of imidazole and 1-phenylimidazole were lower in the absence of exogenous BH4, and both inhibitors also competitively inhibited the BH4-dependent activation of the enzyme. These data taken together suggest that the L-arginine, dioxygen, and the BH4 binding sites are in close proximity in rH-iNOS. Furthermore, these studies demonstrate the usefulness of imidazole compounds as active site probes for recombinant human iNOS.

The Proximal Hydrogen Bond Network Modulates Bacillus subtilis Nitric-oxide Synthase Electronic and Structural Properties

“…The NOS Raman bands were assigned following previous assignments on NOS (46,(55)(56)(57)(58)(59)(60). 66 bsNOS Mutants-The sulfur atom of NOS-proximal thiolate is naturally engaged in a strong H-bond interaction with the nitrogen proton of the indole ring of the vicinal tryptophan (see Scheme 1). In order to check the influence of this H-bond interaction on the proximal thiolate properties, we mutated the bsNOS Trp 66 residue into five different amino acids (see "Experimental Procedures"): a phenylalanine (W66F) and a tyrosine (W66Y) to maintain the aromatic ring but remove the H-bond; a histidine (W66H) that is believed to maintain and even strengthen this H-bond (52); a leucine (W66L) to remove both the H-bond interaction and the aromaticity, keeping some hydrophobic environment; and an alanine (W66A), to remove all possible interactions.…”

Interactions of the α-Spectrin N-terminal Region with β-Spectrin

“…The amount of 125 I-labeled peptide remaining in the wells (cpm bound ) was measured and found to decrease with increasing concentrations of unlabeled peptides ([␣]). A displacement curve was obtained, and the values of IC 50 (concentration of unlabeled peptide needed to inhibit/ displace 50% of the labeled peptide from binding the ␤-spectrin) were determined according to the equation (35,36),…”

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

“…Moreover, in enzyme kinetic studies, H % Bip did not alter the IC &! of NO # Arg for inhibiting the enzyme activity of human pterin-free NOS-II [40]. The reasons for this discrepancy are unclear but might reflect isoform differences or, alternatively, methodological differences, e.g.…”

“…The reasons for this discrepancy are unclear but might reflect isoform differences or, alternatively, methodological differences, e.g. the use of H % Bip-free NOS and measurements under catalytic [40] compared with non-catalytic conditions used in radioligand binding assays [35][36][37][38][39]. There is therefore uncertainty about the functional significance of allosteric binding site interactions in regulating NOS activity and NO generation.…”

“…Allosteric modulation within the catalytic centre of NOS by H % Bip has also been reported [35][36][37][38][39][40]. In a radioligand binding study, Mayer and co-workers demonstrated a 6-fold increase in the affinity of NOS-I for [$H]H % Bip in the presence of -arginine [35].…”