The long term goal of the proposed research is to understand at the molecular level how enzymes achieve their extraordinary catalytic efficiencies. We seek this understanding through direct observation of ligands (substrates, intermediates, and ground-state and transition-state analog inhibitors) bound to enzyme active sites using techniques (primarily NMR and FTIR spectroscopy) sensitive to local bond distortions, electronic environment, and other factors likely to reflect the enzyme's catalytic strategy. We will study the effects of structural alterations in substrates or their analogs and in the enzyme itself. Structural changes in the enzyme will be made using newly developed protein engineering methodology. In addition we shall make whatever kinetic and equilibrium measurements are required for proper interpretation or deeper understanding of the spectroscopic data. We have chosen two groups of enzymes, the Claisen enzymes and the nucleoside aminohydrolases for special emphasis because efficient structural and kinetic data are available to suggest what catalytic strategies be used by these enzymes and our past work shows that intermediate structures characteristic of those strategies can be directly observed. Enzymes which catalyze Claisen condensations are prominent in pathways requiring carbon-carbon bond formation in he biosynthesis of fats and cholesterol (thiolase(s), HMG-CoA synthase) as well in the energy-yielding pathways of glycolysis (citrate synthase) and the malate-isocitrate cycle malate synthase). These enzymes operate through activation of a substrate carbonyl together with stabilization of the alpha-carbanion of the acylthioester cosubstrate. Enzymes which catalyze amino- nucleoside(tide) hydrolysis have more varied but no less vital oles. Adenosine deaminase is necessary to the integrity of the immune response. Other enzymes of this class participate in salvage pathways of purines and pyrimidines guanase, etc) or are involved in the maintenance of cellular energy balance (adenylic deaminase). These enzymes operate through stabilization of tetrahedral intermediate, an analytic strategy used by several other important enzyme groups. Our work will first concentrate on citrate synthase and adenosine deaminase since a great deal of progress as already been made in understanding these enzymes and then our focus will shift to elected other systems within the two groups to expand the context within which we may assess the general applicability of our conclusions. A detailed understanding of metabolic processes at the most fundamental level (how enzymes work) cannot help but lead to a better understand of disease and how to combat it.
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