Phosphoryl transfer is the most common biological reaction. In principle, protein kinases and phosphatases should be excellent targets for drug therapy. There are estimated to be about 3000 protein kinases and phosphatases in the human genome, and these enzymes are central to control of essential cellular processes. However, these enzymes have yet to emerge as powerful drug targets. This proposal is aimed at increasing fundamental understanding of phosphoryl transfer and to probe in depth enzymatic catalysis of these reactions. This understanding can in turn aid in understanding how phosphoryl catalysis is used in complex biological processes and ultimately may contribute to the design of new drugs. This proposal has two sections, the first presenting experiments that probe nonenzymatic phosphoryl transfer reactions and the second section describing experiments with E. coli alkaline phosphatase (AP) that probe basic issues of phosphoryl transfer catalysis. The nonenzymatic studies will provide a fundamental characterization of the reactions of alkyl phosphates. Alkyl phosphates are the most common class of biological phosphate compounds, but the vast majority of studies of phosphate compounds have used aryl for ease of measurement. However, recent results showing unexpectedly fast hydrolysis of methyl phosphate have called into question whether aryl phosphates provide an appropriate model for alkyl phosphates. This will be tested, and the alkyl phosphate reactions characterized, using classical approaches of physical organic chemistry such as linear free energy relationships and also vibrational spectroscopy. Potential catalytic effects from removal of water will be tested and the origin of enhanced hydrolysis in mixed organic solvents will be investigated. E. coli AP is an ideal enzyme to probe fundamental issues in enzymatic catalysis because of the many prior kinetic and mechanistic investigations and the multiple high-resolution structures available, and because the low specificity and open active site allow systematic variation of substrates. AP catalyzes the hydrolysis of phosphate diesters in addition to its cognate reaction with phosphate monoesters. Catalysis of these reactions that proceed through different transition states in solution will allow the basic question to be asked using linear free energy relationships and isotope effects: Does the enzyme or the intrinsic properties of the substrate determine the nature of the reaction's transition state? The active site features responsible for 'tuning' AP for preferential hydrolysis of phosphate monoesters will be tested by site-directed mutagenesis.
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