The dynamic interaction of an enzyme with its substrate occurs along a well-defined catalytic pathway that consists of multiple, elementary steps. As this pathway is traversed, transient enzyme-substrate (E . S) intermediates are formed. In order to fully understand the mechanism of enzyme catalysis, it is essential that the structures of both the enzyme and substrate in each of these intermediates be elucidated. In the past, studies of E . S intermediates have proven difficult because of their brief lifetime andlow concentration. Furthermore, general methods capable of providing information about structure in solution have not been developed. The proposed research combines two areas of current research to overcome these problems. Subzero temperatures and cryosolvents will be used to accumulate intermediates in high concentrations for periods of time that permit their spectroscopic study. The spectroscopy will then be used to study intermediates in which either the substrate or enzyme is chromophoric to yield vibrational spectra of parts of the active site. This research will focus on metalloenzymes because they often have chromophoric centers that are ideally suited for resonance Raman studies. In one series of experiments, the rR spectra of intermediates in the hydrolysis of chromophoric peptide and dithioester substrates by leucine amino-peptidase will be studied to detect structural changes in the substrate during catalysis. In other experiments, rR spectra of intermediates formed during the oxidation of colorless substrates by the chromophoric enzyme horse radish peroxidase will be examined to detect changes in the structure of the heme group as it participates in catalysis.
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