ENZYME EFFECTOR DESIGN

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The hypothesis will be tested that the catalytic power of enzymes originates in biologically co-evolved interactions between enzyme-derived and substrate-derived structures specific to the catalytic transition state interactions which are either absent or weakened in reactant and intermediate states. These actions should be strongest with the "evolutionarily anticipated" enzyme/substrate combinations and disabled to some extent with "unnatural" substrates or mutant enzymes. Enzymes from three classes will be studied: serine proteases, pyruvate decarboxylase and lactate dehydrogenases. Proton inventories with substrates of increasing peptide chain length will be determined to test for multiproton catalysis by the Asp-i02-Asn mutant of trypsin, which will reveal whether the multiproton catalysis is or is not produced by the charge-relay system. Depending on the results, other mutant trypsins may also be studied. The temperature dependence of the solvent isotope effect for chymotrypsin, trypsin, elastase and subtilisin will be measured to deduce the degree to which "physical" steps and "chemical" steps determine the rate, and to probe for the importance of quantum tunneling in enzymic acid-base catalysis. With pyruvate decarboxylase, trapping of the carbanion intermediate will be used to determine intrinsic isotope effects for the decarboxylation step and an accurate free energy diagram will be constructed. The interaction with the enzyme of the substrate-like inhibitors HCOCOOH and CO(COOH)2 will be studied kinetically and by solvent isotope effects. Lactate dehydrogenases from psychrophilic, mesophilic and thermophilic bacteria will be studied by primary and secondary isotope-effect methods to determine how the amino-acid sequence changes arising from thermal adaptation are reflected in transitionstate structure and in the relative rats of individual steps. Solvent isotope effects will probe the importance of electrophilic catalysis of hydride transfer and substrate isotope effects the role of tunneling. A mutant of the thermophilic enzyme will also be examined. The results of these studies should eventually be useful in designing effectors to inhibit or activate enzymes.