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

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• 作者：Renee M. Chabin ; Ermenegilda McCauley ; Jimmy R. Calaycay ; Theresa M. Kelly ; Karen L. MacNaul ; Gloria C. Wolfe ; Nancy I. Hutchinson ; Sayyaparaju Madhusudanaraju ; John A. Schmidt ; John W. Kozarich ; and
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：July 23, 1996
• 年：1996
• 卷：35
• 期：29
• 页码：9567 - 9575
• 全文大小：418K
• 年卷期：v.35,no.29(July 23, 1996)
• ISSN：1520-4995

文摘

Nitric oxide synthase catalyzes the pyridine nucleotide-dependentoxidation of L-arginine tonitric oxide and L-citrulline. It is a specializedcytochrome P₄₅₀ monooxygenase that is sensitive toinhibitionby imidazole. Steady-state kinetic studies on recombinant humaninducible nitric oxide synthase (rH-iNOS) demonstrate that imidazole and 1-phenylimidazole are competitiveand reversible inhibitors versusL-arginine. Structure-activity relationship and pHdependence studies on the inhibition suggest that theneutral form of imidazole may be the preferred species and that theonly modifications allowed withoutthe 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 differencespectra exhibiting K_d values of 63± 2 and 28 ± 3 mages/entities/mgr.gif">M, respectively. These values were in goodagreement with the steady-state K_i of95± 10 and 38 ± 4 mages/entities/mgr.gif">M, respectively, and confirms the site ofbinding is at the sixth axial ligand of theheme. Imidazole (2.2 mM) also perturbed theK_d of L-arginine from 3.03 ± 0.45to 209 ± 10 mages/entities/mgr.gif">M. Theobserved increase in the K_d forL-arginine is consistent with imidazole being a competitiveinhibitor versusL-arginine. The IC₅₀ values of imidazoleand 1-phenylimidazole were lower in the absence ofexogenousBH₄, and both inhibitors also competitively inhibited theBH₄-dependent activation of the enzyme.Thesedata taken together suggest that the L-arginine, dioxygen,and the BH₄ binding sites are in closeproximityin rH-iNOS. Furthermore, these studies demonstrate the usefulnessof imidazole compounds as activesite probes for recombinant human iNOS.