Active-site structure analysis of recombinant human inducible nitric oxide synthase using imidazole.
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.
Chabin
R M
RM
Department of Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
McCauley
E
E
Calaycay
J R
JR
Kelly
T M
TM
MacNaul
K L
KL
Wolfe
G C
GC
Hutchinson
N I
NI
Madhusudanaraju
S
S
Schmidt
J A
JA
Kozarich
J W
JW
Wong
K K
KK
D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000378:metabolism / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D019335:Nitroarginine; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D007093:Imidazoles; D007527:Isoenzymes; D011994:Recombinant Proteins; D019335:Nitroarginine; D001708:Biopterins; C037738:1-phenylimidazole; C029899:imidazole; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin