NOVEL KINETIC TECHNIQUES TO STUDY HYDRATASE REACTIONS

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The reaction mechanism of enoyl-CoA hydratase will be intensively studied by several novel kinetic techniques. The emphasis in these studies is two-fold: to determine if enzymes can convert the specific enzyme-substrate binding energy into catalytic efficiency by facilitating proton transfer reactions and to determine if noncovalent intermediates are significantly stabilized by hydratases during the course of the catalyzed reaction. Both of these experimental goals require the development of new and more precise kinetic techniques which should be generally applicable to other classes of enzymes. The pH variation of the kinetic parameters of crotonase will be examined to determine the importance of the protonation state of the enzyme, the effect of substrate binding on the pK-a-s of active site functional groups and the relative rate of proton transfers in the enzyme-substrate complex. Two proven kinetic techniques, developed for their precision, are adapted to the study of enzyme reactions to provide data of sufficient precision. These techniques should allow us to view how enoyl-CoA hydratase converts binding energy to catalytic efficiency through general acid-base catalysis. The presence of enzyme-bound intermediates that would be too unstable to exist in aqueous solution, like the enol of a carboxylic acid, is an unresolved question. A series of kinetic isotope effects on V-max/K-m will be measured under conditions that one of the bond breaking steps is rate determining to establish the structure of the transition state. By developing methods so that heavy atom and secondary deuterium isotope on V-max can be routinely measured, the relative stability of the different enzyme-substrate complexes will be determined. Direct evidence for the intermediates suggested by the kinetic studies will be search for by quenching the enzyme-substrate complex under conditions that will chemically differentiate any of the substrate that was bound as a reactive intermediate and from spectroscopic studies on the dithio-CoAester substrate analog. The enhanced understanding of enzyme reaction mechanisms contributes to all of the biological sciences. The increased understanding of the role of general acid-base catalysis should lead to improved design of enzyme inhibitors that take advantage of the potential proton transfers and should increase the understanding the effects of alterations at enzyme active sites, bringing us a step closer to the point where we can design enzymes with desired activities.