Serine proteinase inhibitors (serpins) in general, and antichymotrypsin in particular, play a central role in a wide variety of physiologic processes involved in aging. This is especially evident in diseases that increase as a function of age including certain cancers, cardiac and lung diseases and degenerative diseases of the central nervous system and neuromuscular system. These processes critically depend on the balance of proteinases and proteinase inhibitors, a balance that is ultimately determined by the molecular properties of the reaction between the two proteins. The mechanism that determines whether a serpin is an inhibitor, a substrate or shows a stoichiometry of inhibition of greater than one for a given proteinase target is a function of the relative rates of: (1) establishing inhibitor P1 to enzyme S1 site:site interaction and perhaps other subsite interactions, which correspond to partial or full unwinding of the helical portion of the serpin reactive loop; (2) formation of a covalent, metastable acyl-enzyme/inhibitor complex; (3) partial or full insertion of strand 4A, derived from the reactive center loop of the serpin into the serpin betaA sheet; (4) transfer of the energy associated with the conformational change of the serpin into a conformational change in the enzyme that disables the catalytic mechanism; and (5) deacylation of the complex and release of cleaved inhibitor. The applicant proposes to fully elucidate the overall sequence of events leading to metastable acyl-enzyme/inhibitor complex formation and breakdown through stopped-flow and conventional kinetic analyses allied with fluorescence energy transfer and X-ray crystallographic structure determination. In these studies the applicant will employ cloned and recombinantly expressed wild-type and reengineered serine proteinases, chymotrypsin and prostate-specific antigen, and the serpin antichymotrypsin, and fluorescent derivatives of these proteins. These modified proteins will permit determination of the contribution of each residue or combinations of residues of interest to the functional properties of the enzyme and the serpin, focusing on each of the mechanistic steps listed above. The applicants also will pursue studies on the nature of the unique DNA binding properties of antichymotrypsin. The applicant will generate a randomer library and use immobilized ACT to select and PCR amplify specific DNA target sequences for ACT. In addition, the applicant will investigate the formation of hetromeric protein/ACT complexes using the yeast two-hybrid system.
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