The Energetics of Enzyme Catalysis

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This work is aimed at a deeper understanding of the nature of enzyme catalysis. Two groups of enzymes will be studied. First, the mechanism of amino acid racemases will be investigated, with particular focus on glutamate racemase and diaminopimelate empimerase. Each of these enzymes catalyzes the racemization of an x-amino acid center without the need for or involvement of pyridoxal phosphate. The enzymes will be isolated and characterized, and the energetics of their reactions will be explored by the use of isotope exchange, isotope tracer, and perturbation experiments. The second group of enzymes are the hydratases, and the primary goal here will be to determine whether the reactions proceed in a concerted or stepwise fashion. The double isotope fractionation test will be used, and a new protocol will be evaluated that has the potential for extending this methodology to previously inappropriate systems. Hydrations that proceed with anti stereochemistry will be compared with those that follow a syn pathway. Together, the studies proposed will provide information about the intimate mechanistic details of these very efficient reactions. Enzymes are formidably efficient catalysts that mediate specific chemical transformations at rates rarely approached by even the best of man-made catalytic assemblies. If we could trace the development of catalytic efficiency we should be better able to understand the nature of enzyme catalysis as we find it today. Dr. Knowles is certainly making headway with his incisive attack on simple systems.