The strategic goals of this research are to define the structure and interactions of substrate carbonyl groups in enzyme active sites and to relate these to the enzyme's mechanism and reactivity. One class of reactions involves acyl-enzyme intermediates of cysteine proteases, where four of the enzymes are mammalian or viral proteases implicated in disease states and are thus targets for knowledge-based drug design. The second class of reactions features an environmentally important enzyme, 4- chlorobenzoyl-CoA dehalogenase, which participates in the dechlorination of potentially harmful chlorinated hydrocarbons. In all cases, enzyme- carbonyl interactions are a key part of enzyme mechanism and in all cases, Raman spectroscopy offers a unique and powerful means of characterizing the carbonyl's chemistry and environment. For the cysteine proteases the specific aims are: * For the mammalian proteases, cathepsins B and L (together with the plant enzyme papain) and the hepatitis viral protease HAV-3C, and their protein- engineered analogs, to use the carbonyl stretching feature in the Raman difference spectrum of acyl-enzymes to develop precise and novel structure-reactivity relationships. * For the calcium activated cysteine protease, calpain II, to use the Raman spectrum of the bound substrate to probe the effects of calcium activation on the active site. This will provide some of the first molecular information on how calcium regulates the activity of calpain. * For cysteine protease-aldehyde transition state analog complexes, to use Raman difference spectroscopy to obtain the vibrational spectrum of the putative tetrahedral center. Interpretation of these data will provide structural informational on a transition state - like species at the point of catalytic transformation. All these studies will be supported by detailed conformational analyses and vibrational calculations on model compounds and their isotopically substituted analogs, as well as by substrate synthesis and enzyme kinetics. For reactions involving 4-chlorobenzoyl-CoA dehalogenase the specific aims are: * To use the Raman data obtained from the enzyme-bound product, 4- hydroxybenzoyl-CoA, and various substrate analogs to provide some of the first detailed molecular information on the large electron rearrangements occurring in the substrate along the reaction pathway and how these changes are mediated by benzoyl carbonyl - active site interactions.
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