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 subfamily, including rat P-450 2B1, 2B1-WM (an allelic variant of 2B1 expressed in Wistar-Munich rats), 2B2, and rabbit 2B4 and 2B5. The major products formed with all isoforms were the four regioisomeric epoxides, but 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-450 2B2, 2B4, and 2B5 catalyzed the formation of all four epoxides in nearly equal amounts. The single Gly-478-->Ala substitution in the variant P-450 2B1-WM was sufficient to cause a dramatic change in the ratio of epoxides when compared with P-450 2B1. The Gly-478-->Ala mutation also changed the regioselective epoxidation of gamma-linolenic acid at the three double bonds. Four site-directed mutants of P-450 2B1 were also evaluated. The mutations included two single mutants where Ile-114 was changed to either Val or Ala and two double mutants where the 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 arachidonic acid at all four double bonds was nearly equal. However, substitution of Ile-114 with Ala, resulted in a significant reduction in the degree 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 position 478 in conjunction with Val at position 114 the regioselective epoxidation 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 epoxide 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.
Laethem
R M
RM
Department of Pharmacology, Oregon Health Sciences University, Portland 97201-3098.
Halpert
J R
JR
Koop
D R
DR
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D051381:Rats; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D017962:alpha-Linolenic Acid / Q000378:metabolism
D004852:Epoxy Compounds; D007527:Isoenzymes; D017962:alpha-Linolenic Acid; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase