ISOTOPIC PROBES OF ENZYMATIC REACTION MECHANISMS

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The broad, long-term objectives for the research described in this proposal are aimed at the elucidation of the complex relationships between structure and function in biological systems. The primary focus of the present application is directed at the mechanism of catalysis and structure of the bacterial phosphotriesterase. This enzyme catalyzes the detoxification or organophosphate neurotoxins through the hydrolysis of phosphorus-oxygen and phosphorus-fluorine bonds. The phosphotriesterase catalyzes the hydrolysis of optimal substrates at the diffusion- controlled limit and a divalent cation is required for catalytic activity. The role of the essential metal ion will be probed by substitution of the native Zn2+ ion with a variety of spectroscopically active cations (Cd2+, Mn2+, Ni2+, Co2+, and Cu2+). The ligand environment of the metal sites will be addressed by 113Cd-NMR spectroscopy. Interactions between the metal sites and substrates will be obtained using ESR spectroscopy with the paramagnetic metal ion derivatives. The spectroscopic studies will be complemented by replacement of potential amino acid ligands (histidine, cysteine, aspartate, and glutamate) using site-directed mutagenesis protocols. The mutant proteins will be utilized for the sequence specific assignments of the protein ligands to the metal centers and as probes for the role these metal ions play in the catalytic events. The identity and function of other amino acids at the active site of this enzyme will be determined through the design and synthesis of alkynyl phosphate esters. These suicide substrates covalently react with active site nucleophiles and result in complete inactivation of catalysis. Heavy atom oxygen-18 isotope effects with fast and slow substrates using wild type and mutant proteins will be utilized to determine the distribution of transition state structures. An X-ray crystallographic analysis will begin in an effort to determine the three-dimensional structure of the folded protein.