ENZYME EFFECTOR DESIGN

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The catalytic power of serine proteases, amidohydrolases from various bacteria, pyruvate decarboxylase, and lactate dehydrogenases from a psychrophile, a mesophile and a thermophile, GABA-T and possibly some other enzymes will be investigated through the determination of primary and secondary hydrogen isotope effects, carbon isotope effects, and solvent isotope effects. The latter will be analyzed by the proton-inventory method to dissect the overall effect into contributions from individual sites, wherever possible. The objective is to learn from the isotope effects about the structure of the transition states for the enzymic reactions and to compare the enzymic transition states with those for other enzymic reactions and non-enzymic reactions, in order to deduce how the enzyme is producing its catalytic acceleration. Isotope effects, by defining the nature of the steps which contribute to limiting the rate under various conditions, also help to show how enzymes combine multistep processes to generate a complete mechanistic sequence. For the serine proteases, proton inventories will be conducted with oligopeptide substrates of various length and sequence in order to discover what the prerequisites are for coupling the multiproton catalytic machinery of the enzyme. The degree to which substrate structural changes at the reacting carbonyl accompany this activation will be investigated with secondary deuterium isotope effects. Similar experiments with various substrates of asparaginases and glutaminases will be carried out. For pyruvate decarboxylase, the effects of pH and temperature on directly measured C-13 effects will be used to discover how these variables affect the contribution of decarboxylation and other steps to rate limitation. Solvent isotope effects will be similarly used to probe the role of proton-transfer components of the mechanism. For lactate dehydrogenases, primary and secondary hydrogen effects should reveal how the changes in amino-acid sequence which have accompanied the genetic adaptation of the bacteria to different temperature regimes are reflected in the structure of the activated complexes and the relative contributions of different steps to rate limitation, both under optimal and non-optimal conditions for each enzyme. Temperature dependences of solvent isotope effects for hydrolytic enzymes will also test whether a tunneling model is required for enzymic acid-base catalysis.