TY - JOUR
T1 - Active-site structure analysis of recombinant human inducible nitric oxide synthase using imidazole
AU - Chabin, Renee M.
AU - McCauley, Ermenegilda
AU - Calaycay, Jimmy R.
AU - Kelly, Theresa M.
AU - MacNaul, Karen L.
AU - Wolfe, Gloria C.
AU - Hutchinson, Nancy I.
AU - Madhusudanaraju, Sayyaparaju
AU - Schmidt, John A.
AU - Kozarich, John W.
AU - Wong, Kenny K.
PY - 1996
Y1 - 1996
N2 - 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 (rHiNOS) 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 rHiNOS with imidazole and 1-phenylimidazole yielded type II difference spectra exhibiting K(d) values of 63 ± 2 and 28 ± 3 μM, respectively. These values were in good agreement with the steady-state K(i) of 95 ± 10 and 38 ± 4 μM, respectively, and confirms the site of binding is at the sixth axial ligand of the heme. Imidazole (2.2 mM) also perturbed the K(d) of L-arginine from 3.03 ± 0.45 to 209 ± 10 μM. The observed increase in the K(d) 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.
AB - 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 (rHiNOS) 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 rHiNOS with imidazole and 1-phenylimidazole yielded type II difference spectra exhibiting K(d) values of 63 ± 2 and 28 ± 3 μM, respectively. These values were in good agreement with the steady-state K(i) of 95 ± 10 and 38 ± 4 μM, respectively, and confirms the site of binding is at the sixth axial ligand of the heme. Imidazole (2.2 mM) also perturbed the K(d) of L-arginine from 3.03 ± 0.45 to 209 ± 10 μM. The observed increase in the K(d) 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.
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U2 - 10.1021/bi960476o
DO - 10.1021/bi960476o
M3 - Article
C2 - 8755738
AN - SCOPUS:0029740456
SN - 0006-2960
VL - 35
SP - 9567
EP - 9575
JO - Biochemistry
JF - Biochemistry
IS - 29
ER -