The dynamic interaction of an enzyme (E) with its substrate(S) occurs along a well-defined catalytic pathway that consists of multiple, elementary steps. As this pathway is traversed, transient enzyme-substrate intermediates, ESi are formed. In order to fully understand the mechanism of enzyme catalysis, it is essential that these. ES1 be detected and that their relative energies, rates of interconversion and structures be determined. Such studies are inherently difficult because of the brief lifetime and low concentration of such species. In the proposed research, a new low temperature stopped- flow instrument will be constructed and stopped-flow cryoenzymology will be used to study the pathway for the hydrolysis of dansyl-peptide substrates by thermolysin. The use of subzero temperatures and cryosolvents will slow down the rate of interconversion of ESi along the pathway. Radiationless energy transfer between tryptophan residues of the enzyme of the enzyme and the dansyl group of the substrate will be used to visualize the ESi. The rate constants and activation energies for each elementary step in the reaction will be determined. Thermolysin is a zinc proteinase that retains activity when Co(II), Mn(II) and Fe(II) are substituted at the active site,and studies will be carried out with all four species to investigate the role of the metal ion in each step. A new rapid scan diode array detector will be constructed and rapid scan optical studies will be carried out with Co(II)-thermolysin to characterize the structure of the metal center in each ESi.
