This grant from the Organic Dynamics Program supports the work of Dr. Donald Hilvert at the Scripps Research Institute to study physicochemical and structural characterization of selenosubtilisin with particular emphasis on understanding and optimizing its peptide ligase and peroxidase activities. Subtilisin variants containing site-specific changes in addition to Ser221Sec will be prepared to address mechanistic questions and to improve the chemical efficiency of the first-generation catalysts. The specific targets are (a) to prepare a family of artificial selenoenzymes based on subtilisin and explore the intrinsic reactivity of its selenium prosthetic group, (b) to characterize these selenocysteine-containing proteins and deduce the relative importance of steric and electronic factors in catalysis, (c) to apply them to peptide ligation, (d) to characterize and optimize their redox properties to both mimic the action of the natural redox enzyme glutathione peroxidase and catalyze the stereospecific oxidation of substrates other than thiols, and (e) to extend these studies to other protein templates. In this project Dr. Hilvert will elaborate structural, mechanistic and synthetic aspects of selenoenzymes prepared by modifying naturally occurring enzymes, where the modification entails a replacement of sulfur (or oxygen) by selenium. The work concerns the preparation and characterization of selenosubtilisin and variants thereof, peptide ligation, redox chemistry, and expansion of selenium substitution in other enzymes. The rational design of these abzymes may have a profound impact on medicinal and industrial applications.
