We wish to explore the molecular mechanisms and regulation of important physiological processes catalyzed by metalloenzymes. in the present grant period we will focus on design, synthesis, and characterization of mechanism-based inhibitors of steroid hydroxylases. This work is based on our finding of four types of effective inhibitors of Cytochrome P-450scc, which catalyzes the six-electron oxidative cleavage of cholesterol to pregnenolone and isocaproaldehyde. These inhibitors depend on oxygen insertion in alkynyl steroids to yeild reactive oxirenes, addition to thioethers to form tight binding sulfoxides, abstraction of hydrogen frm trimethylsilylethyl groups to lead to reactive radical or carbocation groups, and tight binding competitive inhibition by amines of correct stereochemistry. We will now characterize the geometry of the P450scc active site by EXAFS and Electron Spin Echo modulation spectroscopies, and study the localizations and fate of the inhibitors in whole adrenal cells and in animals. We will make and test other inhibitors specific to the other steroid exygenases, and study their effect on estrogen, androgen, and mineralocorticoid formation . We will also continue our fundamental work on electron transfer proteins' structure and kinetics. The current period of the project may see the emergence of substances useful in the therapy of steroid-dependent neoplasia.
