96-04379 Craik Part 1. Technical The study is to provide insight into the substrate specificity, catalytic mechanism, and structure of the chymotrypsin family of serine proteases. Variant enzymes will be generated using current methods of protein engineering. Two general approaches are used to alter the enzyme: (1) targeted substitutions followed by analysis of the variant enzyme; and (2) random substitutions coupled with an in vitro affinity selection. In each case, variants of serine proteases are generated according to structural and/or functional principles to understand the specificity and catalytic power of the enzyme and use this understanding to alter the activity of the enzyme in a predictable fashion. Variant enzymes are designed, isolated, and purified, and their kinetic parameters compared with the native enzyme. Selected variants are then chosen for more detailed kinetic, theoretical, and three-dimensional structure analysis. Results form the inital modifications will provide a basis for introducing future amino acid changes into the enzyme. Initial experiments have been successful in addressing the role of specific amino acids in substrate recognition and catalytic mechanism of the enzyme trypsin and in using that information to alter the enzyme activity using designed metal binding sites and substrate assisted catalysis. Other experiments involve amino acid replacements for studies including substrate recognition and metalloregulation. Variant proteases are designed by computer modeling based on our current understanding of proteases including fiddler crab collagenase, granzymes, and other related members of this family. These studies will contribute to our understanding of structure/function relationships in serine proteases. In addtion, these studies will help develop unique specificities for peptide and protein hydrolysis. Part 2. non-technical The relationship between the three dimensional structure and the function of an enzyme is still poorly understood. This is particularly evident in the field of protein engineering where efforts are made to alter the activity of a protein in a predictable fashion. Macromolecular recognition and the basis of catalytic power in an enzyme remain unexplained in terms of basic general principles that can be applied to a protein catalyst. This study will address these two fundamental questions as a long term goal by studying the contributions of certain amino acids in the substrate specificity and catalytic mechanism of serine protease. The aim is to be achieved by creating and analyzing altered protease which contribute to our understanding of mechanisms involved in ligand binding and bond making and bonding breaking. The altered enzyme will be generated using current methods of protein engineering as designed by computer modeling based on our current knowledge of proteases of this family. These studies will contribute to our understanding of structure/function relationships in protein degradative enzymes. ***
