Reconstitution of the second step in NO synthesis using the isolated oxygenase and reductase domains of macrophage NO synthase

Biochemistry
D K GhoshD J Stuehr

Abstract

Inducible macrophage NO synthase (iNOS) is a homodimer of 130 kDa subunits. Trypsinolysis of iNOS inactivates its NO synthesis activity and cleaves the enzyme into a dimeric oxygenase fragment that contains heme, tetrahydrobiopterin, and the substrate binding site and a monomeric reductase fragment that contains FAD, FMN, calmodulin, and the binding site for NADPH [Ghosh, D. I., & Stuehr, D. H. (1995) Biochemistry 34, 801-807]. In this paper, we describe the reconstitution of NO synthesis activity utilizing the isolated oxygenase and reductase domains of iNOS. Mixing the domains at various ratios showed that NO was not produced from L-arginine but could be formed from the reaction intermediate N omega-hydroxy-L-arginine (L-NOHA). The apparent Km with L-NOHA in the reconstituted system was 100 microM versus 19 microM for native iNOS. D-NOHA was not a substrate. Maximum specific activity (per heme) occurred at an oxygenase to reductase molar ratio of 4:1, with higher ratios causing some inhibition. Reconstitution of activity was associated with electron transfer between the domain fragments and led to an incomplete reduction of the oxygenase domain heme iron. L-NOHA, but not L-arginine, increased NADPH consumption in the reconsti...Continue Reading

Citations

Apr 1, 1997·Protein Science : a Publication of the Protein Society·M A ShermanM T Mas
Mar 18, 2009·Biochemistry·Robielyn P IlaganDennis J Stuehr
Jun 7, 2012·The Journal of Physical Chemistry. a·Andrei V AstashkinChangjian Feng
Jul 21, 2001·The Biochemical Journal·W K AldertonR G Knowles
Jan 1, 1997·Annual Review of Pharmacology and Toxicology·D J Stuehr
Jan 5, 2002·Proceedings of the National Academy of Sciences of the United States of America·Subrata AdakDennis J Stuehr
Apr 16, 2015·Journal of the American Chemical Society·Wei TangRichard B Silverman
Jun 14, 2003·Biochemical and Biophysical Research Communications·Rajib SenguptaSanjay Ghosh
Jan 5, 1999·The Journal of Biological Chemistry·N SennequierD J Stuehr
Aug 14, 1999·The Journal of Biological Chemistry·S GhoshD J Stuehr
Nov 28, 2006·The Journal of Biological Chemistry·Zhi-Qiang WangDennis J Stuehr
May 13, 1999·Biochimica Et Biophysica Acta·D J Stuehr
Feb 8, 2006·Journal of Theoretical Biology·J Christian J Ray, Denise E Kirschner

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