CLONING, EXPRESSION & CHARACTERIZATION OF GDH MUTANTS

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Mammalian Glutamate Dehydrogenases play an essential role in nitrogen metabolism in the liver and appear to play a vital role in glutamate metabolism in other tissues, especially the brain. Glutamate Dehydrogenase is a complex, allosterically regulated enzyme that in mammalian systems appears to be associated with ammonia metabolism. The proposed work will define structure function relationships involved in this enzyme. The work, in conjunction with the determination of the three-dimensional structure by a collaborating group, will define amino acid side chains involved in substrate binding, in cofactor and allosteric regulator binding, and in the regions of subunit interaction in this hexameric enzyme. The gene for Bovine Glutamate Dehydrogenase will be cloned, and an expression system developed to allow site-directed mutants to be constructed.This will involve using a yeast expression system in order to obtain appropriate processing of this mammalian intramitochondrial protein. Expressed protein will be purified using a novel affinity chromatography system specific for mammalian Glutamate Dehydrogenase. pH dependence and chemical reactivity studies of defined residues will help to elucidate the functional characteristics of residues that are not apparent simply from the structure. This work will form the basis of site-directed mutagenesis approaches and will be conducted in conjunction with the necessary enzyme characterization by kinetic methods as well as structure determination of mutants. Site-directed mutants will be used to examine the role of potential functional residues in the active site, in the ADP regulatory site, and in subunit interactions in this enzyme. The successful completion of this project will give new insights into this complex and important regulatory enzyme, and should provide, for the first time, a clear understanding of both structure-function relationships in the active site of this enzyme, and the structural basis of, and potential role for, negative homotropic interactions in a mammalian Glutamate Dehydrogenase.