EPOXIDATION OF ARACHIDONIC-ACID AS AN ACTIVE-SITE PROBE OF CYTOCHROME-P-450 2B ISOFORMS

In the present study we determined the regioselectivity of arachidonic acid epoxidation by several members of the cytochrome P-450 2B subfam ily, including rat P-450 2B1, 2B1-WM (an allelic variant of 2B1 expres sed in Wistar-Munich rats), 2B2, and rabbit 2B4 and 2B5. The major pro ducts formed with all isoforms were the four regioisomeric epoxides, b ut each isoform produced a distinct distribution of the four epoxides. P-450 2B1 produced predominantly 14,15-epoxyeicosatrienoic acid (EET) , while P-450 2B1-WM produced the 11,12-EET as the major product. P-45 0 2B2, 2B4, and 2B5 catalyzed the formation of all four epoxides in ne arly equal amounts. The single Gly-478 --> Ala substitution in the var iant P-450 2B1-WM was sufficient to cause a dramatic change in the rat io of epoxides when compared with P-450 2B1. The Gly-478 --> Ala mutat ion also changed the regioselective epoxidation of gamma-linolenic aci d at the three double bonds. Four site-directed mutants of P-450 2B1 w ere also evaluated. The mutations included two single mutants where Il e-114 was changed to either Val or Ala and two double mutants where th e Ala-478 mutation was coupled with either Val or Ala at position 114. When Ile-114 was mutated to Val, the degree of epoxidation of arachid onic acid at all four double bonds was nearly equal. However, substitu tion of Ile-114 with Ala, resulted in a significant reduction in the d egree of epoxidation at the 14,15- and 11,12-double bonds, and the 8,9 - and 5,6-EETs were the major products. When Ala was introduced at pos ition 478 in conjunction with Val at position 114 the regioselective e poxidation of the mutant enzyme more closely resembled P-450 2B1-WM in that 11,12-EET was the major metabolite. The double mutation with Ala at both positions 114 and 478 produced a unique distribution of epoxi de products with 5,6-EET as the major metabolite. The results of these studies indicate that residues 114 and 478 in the P-450 2B subfamily are important for the orientation of fatty acids in the active site.