Identification of unique amino acids that modulate CYP4A7 activity

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 SRS 1 and prostaglandin activity is influenced by SRS2-3. Site-directed mutant proteins of CYP4A7 found laurate and arachidonate acti vity markedly diminished in the R90W mutant (SRS1) and somewhat decreased i n W93S. While PGE(1) activity was only slightly increased, the mutant prote ins H206Y and S255F (SRS2-3), on the other hand, exhibited remarkable incre ases in laurate and arachidonate metabolism (3-fold) above wild-type substr ate metabolism. Mutant proteins H206Y, S255F, and H206Y/ S255F but not R90W /W93S, wild-type CYP4A4, or CYP4A7 metabolized arachidonic acid in the abse nce of cytochrome bs. Stopped-flow kinetic experiments were performed in a CO-saturated environment performed to estimate interaction rates of the mon ooxygenase 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 local ization within the enzyme structure based on comparisons with homologous, k nown cytochrome P450 structures.