Identification of Unique Amino Acids That Modulate CYP4A7 Activity

Abstract

A multifamily sequence alignment of the rabbit CYP4A members with the known structure of CYP102 indicates amino acid differences falling within the so-called substrate recognition site(s) (SRS). Chimeric proteins constructed between CYP4A4 and CYP4A7 indicate that laurate activity is affected by the residues within SRS1 and prostaglandin activity is influenced by SRS2-3. Site-directed mutant proteins of CYP4A7 found laurate and arachidonate activity markedly diminished in the R90W mutant (SRS1) and somewhat decreased in W93S. While PGE(1) activity was only slightly increased, the mutant proteins H206Y and S255F (SRS2-3), on the other hand, exhibited remarkable increases in laurate and arachidonate metabolism (3-fold) above wild-type substrate metabolism. Mutant proteins H206Y, S255F, and H206Y/S255F but not R90W/W93S, wild-type CYP4A4, or CYP4A7 metabolized arachidonic acid in the absence of cytochrome b(5). Stopped-flow kinetic experiments were performed in a CO-saturated environment performed to estimate interaction rates of the monooxygenase reaction components. The mutant protein H206Y, which exhibits 3-fold higher than wild-type substrate activity, interacts with CPR at a rate at least 10 times faster than that of wild-type CYP4A7. These experimental results provide insight regarding the residues responsible for modulation of substrate specificity, affinity, and kinetics, as well as possible localization within the enzyme structure based on comparisons with homologous, known cytochrome P450 structures.

Stimulation of Cytochrome P450 Reactions by Apo-cytochromeb 5

“…Because P450 2E1 activities are stimulated by b 5 but not by apo-b 5 (66), a case can clearly be made there for electron transfer. With P450 3A4, both apo-b 5 and b 5 stimulated the reduction of ferric P450 in the presence of the substrate testosterone (22,27); this effect was not observed with P450 2C9 (28) and was observed to only a limited extent with 4A7 (30,31). The K m (testosterone) and oxidation-reduction potential for P450 3A4 were not changed (22).…”

“…1A1, 1A2, 1B1, and 2D6). 2 The conclusion regarding these experiments with apo-b 5 is that electron transfer (to P450) by b 5 is not involved in the stimulation (22,29,31). The effect of b 5 varies depending upon the particular P450 3A4 system.…”

“…We extended this work and reported that P450 2C9 reactions were stimulated by apo-b 5 as well as b 5 , but with P450 2E1, only b 5 was stimulatory (28). Other laboratories reported the stimulation of P450s 17A (29) and 4A7 (30,31) by apo-b 5 . Recent work in our own groups has also shown stimulation of catalytic activities of P450s 2A6, 2B6, 2C8, 2C19, and 3A5 by both b 5 and apo-b 5 .…”

“…Sigma sequencing grade horse apomyoglobin was utilized in our work (this is a standard for optimization of amino acid sequenators). Our work was all done with rabbit b 5 and apo-b 5 instead of human apo-b 5 (39), although the stimulation of P450 activities by b 5 /apo-b 5 has been demonstrated with proteins from varying sources (22,29,31), and the difference is not expected to account for the varying results.…”

Cytochrome P450 7A1 Cholesterol 7α-Hydroxylation

“…However, it was noted (45) that even this fit is precarious in that the P450 7A1 mutant A358V formed 7␤-OH cholesterol and an unidentified product, and we have recently determined that 7-dehydrocholesterol, the immediate precursor of cholesterol, is also a substrate for P450 7A1. 6 In summary, we evaluated the kinetic mechanism of human P450 7A1-catalyzed cholesterol 7␣-hydroxylation, measuring rate constants of individual steps and kinetic deuterium isotope effects. A minimal kinetic model for the P450 7A1 reaction indicates that the first electron transfer step is rate-limiting and that this is a clearly different phenomenon compared with other P450s that have much lower rates of catalysis.…”