Abstract
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 γ-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-ll4 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-ll4 was mutated to Val, the degree of epoxidation of arachidonic acid at all four double bonds was nearly equal. However, substitution of Ile-ll4 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 2BI-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.
Original language | English (US) |
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Pages (from-to) | 42-48 |
Number of pages | 7 |
Journal | Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular |
Volume | 1206 |
Issue number | 1 |
DOIs | |
State | Published - May 18 1994 |
Externally published | Yes |
Keywords
- Arachidonic acid
- Cytochrome P-450
- Eicosatrienoic acid
- Heterologous expression
- P-450 2B subfamily
- Site-directed mutagenesis
- Structure-function relationship
ASJC Scopus subject areas
- Biophysics
- Structural Biology
- Biochemistry
- Molecular Biology