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Volume 272, Number 6, Issue of February 7, 1997 pp. 3657-3662
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Prostacyclin Synthase Active Sites
IDENTIFICATION BY MOLECULAR MODELING-GUIDED SITE-DIRECTED MUTAGENESIS

(Received for publication, October 1, 1996, and in revised form, November 13, 1996)

From the Vascular Biology Research Center and Division of Hematology, University of Texas Medical School, Houston, Texas 77030

Prostacyclin synthase (PGIS), a cytochrome P450 enzyme, catalyzes the biosynthesis of a physiologically important molecule, prostacyclin. In this study we have used a molecular modeling-guided site-directed mutagenesis to predict the active sites in substrate binding pocket and heme environment of PGIS. A three-dimensional model of PGIS was constructed using P450_BM-3 crystal structure as the template. Our results indicate that residues Ile⁶⁷, Val⁷⁶, Leu³⁸⁴, Pro³⁵⁵, Glu³⁶⁰, and Asp³⁶⁴, which were suggested to be located at one side of lining of the substrate binding pocket, are essential for catalytic activity. This region containing 1-1, 1-2, 1-3, and 1-4 strands is predicted well by the model. At the heme region, Cys⁴⁴¹ was confirmed to be the proximal axial ligand of heme iron. The conserved Phe and Arg in P450_BM-3 were substituted by Leu¹¹² and Asp⁴³⁹, respectively in PGIS. Alteration of Leu¹¹² to Phe retained the activity, indicating that Leu¹¹² is a functional substitution for Phe. In contrast, mutant Asp⁴³⁹  Ala exhibited a slight increase in activity. This result implies a difference in the heme region between P450_BM-3 and PGIS. Our results also indicate that stability of PGIS expression is not affected by heme site or active site mutations.

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