This research proposal will focus on the determination of molecular and kinetic properties of four members of the CYP4A gene subfamily of rabbit cytochromes P450 that hydroxylate medium and/or long chain fatty acids and eIcosanoids primarily or exclusively in the omega-position. Cytochromes P4504A4, 4A5, 4A6 and 4A7 share 85% sequence identity at the amino acid level, yet their substrate specificities vary greatly. The physiological functions of these enzymes remain unclear but abundant literature documents their roles in hemodynamic regulation by controlling vascular tone. Cerebral and renal microvessels are contracted by omega-hydroxyarachidonic acid at concentrations of <10-10 M, for example. In the previous funding period these enzymes were cloned and expressed in E. coli using techniques unique to each isoform. It is now possible to prepare adequate quantities for experiments leading to the understanding of their functions. The generation of these closely related family members has permitted comparisons among them with respect to amino acid differences that could account for their substrate preferences. Also, a primary interest is the determination of the interactions of cytochrome b5 with these enzymes, which result in the marked activation of omega-hydroxylation activities. In order to understand these interactions, the following Specific Aims are proposed: 1) interactions of CYP4As with Substrate: Using the aforementioned expression systems, additional mutations of the various CYP4As wilt be constructed to compare the roles of unique, homologous and, in some cases, identical residues on the activities of the resulting proteins with respect to substrate specificity. The binding constants and rates of binding of various substrates to the CYP4As will be examined by stopped-flow spectrophotometry in the absence and presence of NADPHcytochrome P450 reductase and/or cytochrome b5. 2) Interactions of CYP4As with Other Proteins: Surface plasmon resonance (Biacore), stopped flow spectroscopic, Soret CD, and analytical ultracentrifugation methodologies will be used to examine the rates and thermodynamics of the binding of various expressed CYP4A subfamily members with their common redox partner, NADPH-cytochrome P450 reductase, in the presence and absence cytochrome b5. In addition, mutations of putative surface residues on the CYP4A sub-family members will attempt to map the binding sites for these proteins. 3) Electron Transfer/Acceptor Properties of CYP4A Monooxygenase System: The rates of electron flux NADPH-cytochrome P450 reductase and CYP4As will be measured by stopped-flow spectrophotometry at wavelengths specific for the flavins or heme prosthetic groups to determine the effects on electron transfer of cyt b5, using various substrates, specific to each CYP4A.
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