MOLECULAR MODELING OF HUMAN CYTOCHROME P450C17 & P450C21

点击翻译按钮获取中文摘要

Cytochrome P450c17 catalyzes both the 17-hydroxylation and oxidative cleavage of C21 steroids in both human adrenal glands and gonads. Whereas 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. Determining the three-dimensional structure of P450c17 would greatly enhance our understanding of how these two funamentally distinct activities of a single protein can be independently regulated. P450c21 is a related steroidogenic enzyme that catalyzes the 21-hydroxylase reaction. P450c17 and P450c21 have high sequence similarity and identical gene structures, and both utilize the same substrates (pregnenolone, progesterone, and their 17-hydroxy derivatives). The subtle differences in their substrate selectivities and catalytic specificities, however, lead to the production of different steroid hormones with vastly different biological activities. We believe that structure-function comparisons of the two enzymes will yield important insight to the actions of steroidogenic enzymes and approaches to their selective inhibition. We have completed a computer graphic model of human P450c17 based on the x-ray structure of the bacterial P450BM-P. We will now construct a model of P450c21 using this same approach. We have docked steroid substrates into the P450c17 model to confirm that the model predicts the known activities of the enzyme and explains the loss of activity by mutations that occur in patients. We will perform a similar analysis of the P450c21 structure once completed. These models will be tested by site-directed mutagenesis and we will refine the models with these results. Finally, we will identify segments of the two models that appear to confer catalytic specificity, and we will generate chimeric proteins in an attempt to confer the activities of one enzyme upon the other.