PROSTACYCLIN SYNTHASE ACTIVE-SITES - IDENTIFICATION BY MOLECULAR MODELING-GUIDED SITE-DIRECTED MUTAGENESIS

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 mut agenesis to predict the active sites in substrate binding pocket and h eme environment of PGIS. A three-dimensional model of PGIS was constru cted using P450(BM-3) crystal structure as the template. Our results i ndicate that residues Ile(67), Val(76), Leu(384), Pro(355), Glu(360), and Asp(364), which were suggested to be located at one side of lining of the substrate binding pocket, are essential for catalytic activity . This region containing beta 1-1, beta 1-2, beta 1-3, and beta 1-4 st rands is predicted well by the model, At the heme region, Cys(441) was confirmed to be the proximal axial ligand of heme iron. The conserved Phe and Arg in P450(BM-3) were substituted by Leu(112) and Asp(439) r espectively in PGIS. Alteration of Leu(112) to Phe retained the activi ty, indicating that Leu(112) is a functional substitution for Phe. In contrast, mutant Asp(439) --> Ala exhibited a slight increase in activ ity. This result implies a difference in the heme region between P450( BM-3) and PGIS, Our results also indicate that stability of PGIS expre ssion is not affected by heme site or active site mutations.