MUTAGENESIS OF PYRIDOXAL PHOSPHATE-DEPENDENT ENZYMES

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The objectives and specific aims of this research are: 1) To redesign the catalytic specificity of aspartate aminotransferase, an enzyme which transaminates the dicarboxylic amino acids aspartate and glutamate, to that of tyrosine aminotransferase which has a much broader amino acid specificity. Site directed mutagenesis of aspartate aminotransferase will be based on computer modeling of the two homologous structures. 2) In vitro translation will be used to introduce unnatural amino acids at particularly crucial loci in aspartate aminotransferase in order to interject more subtle variations in mechanism and substrate specificity than can be obtained from the standard set of nineteen amino acids obtainable by site directed mutagenesis technology. 3) A deletion of the active site lysine side chain (K258A) will be made in tryrosme aminotransferase. This enzyme catalyzes the transamination of substituted phenyl glycines. It will therefore be possible for the first time to study both classical Bronsted and Hammett relations for the same enzyme catalyzed reactions. The results should provide details about the reaction coordinate surface as well as of the transition state structure. 4) Site directed mutagenesis will be used to explore the hypothesis that Cys191 has been retained in aspartate aminotransferase isozymes, because it was caught in an evolutionary well from which there is no single base change escape. 5) We will use fluorescence spectroscopy and other physical methods to attempt to discover the physical basis for the slow, kinetically competent, (t1/2=10 minutes) conformational change introduced by the mutation D222A, and 7) The active site of amino cydopropane carboxylate synthase (ACC synthase) is virtually 100% conserved in comparison to aspartate aminotransferase. The former enzyme, which is important for the biosynthesis of the plant ripening hormone ethylene, will be heterologously expressed in E. coli or yeast, and the putative active site amino acids mutated. The mutations will be based on our present understanding of the corresponding substitutions in aspartate aminotransferase.