Cytochrome P450c17 catalyzes both 17-hydroxylation and 17,20 oxidative cleavage of C21 steroids in both human adrenal glands and gonads. While adrenal 17-hydroxylase activity remains fairly constant after birth, adrenal lyase activity, reflected by serum dehydroepiandrosterone sulfate, rises during adrenarche at about age 8 and then declines progressively after age 50. Our laboratory has shown that treatment of human fetal adrenal microsomes with alkaline phosphatase abolishes lyase activity without affecting 17-hydroxylation, suggesting that phosphorylation/dephosphorylation regulates P450c17 lyase activity. We will study the structural and functional consequences of P450c17 phosphorylation in detail through an integrated series of enzymologic and computer graphics studies. Since membrane-bound P450 enzymes are not amenable to study by crystallography, structural studies of mammalian P450s have been based on crystal structures of bacterial P450s. Our laboratory has previously used the CGL to construct a P450c17 model based on the structure of P450cam from P putida. Subsequently, the crystal structures of 3 other P450s (-terp, -BMP, and eryF) have shown that, although the coordinates of half the atoms in these proteins differ by 2 - 15 angstroms, all four share a common set of core structural elements. Dr. Julian Peterson and coworkers at Southwestern Medical Center in Dallas extrapolated this core element motif to a model of P450arom based on the assembly of putative core structural elements as a first step. In collaboration with Peterson's group, we have begun to build an analogous model of P450c17. We have identified the amino acids predicted to contribute to the core structural units and have created an initial core structure by substituting amino acid sidechains of c17 for those of P450BM-P in the -BM-P crystal structure. Without any additional manipulation, the core structure places most hydrophilic residues on the exterior of the protein. We will now group these core units by domains and apply energy minimization. We will combine these domains and connect peptide chains with loop structures from protein structure databases before final energy minimization. We will identify residues predicted to selectively participate in hydroxylase or lyase activities and to direct specific steps in the catalytic cycle, such as electron transfer, substrate binding, and oxygenation. We will test these predictions using site-directed mutagenesis to generate specifically altered proteins for enzymologic experiments, and in so doing, we will refine the model in an iterative manner.
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