Glutamate dehydrogenase from vertebrate sources is regulated by GTP which inhibits, ADP which activates and DPNH which inhibits by binding to a site distinct from the active site. In contrast, enzyme from lower organisms is non-allosteric, although all glutamate dehydrogenases appear to have similar catalytic properties. The amino acid sequences of enzymes from these diverse sources is known, but localization of the catalytic and purine nucleotide regulatory sites within the sequences has only recently been initiated. It is proposed to use several adenine and guanine nucleotide analogues synthesized in this laboratory, in addition to derivatives of substrates, as affinity labels of the distinct catalytic and regulatory sites of glutamate dehydrogenase. These compounds have alkylating functional groups capable of reacting with amino acid residues of the enzyme. In the case of each specific modification, the kinetic, binding and physical properties of the altered enzyme will be examined to elucidate its functional aberration. Characterization of the binding sites will be accomplished by isolation of radioactive peptides generated by proteolytic digestion of labeled enzymes. Advantage will be taken of the special properties of nucleoside-linked peptides in their purification. The amino acid composition and sequence of these peptides will be determined and the modified amino acid residues will be identified by comparison with model compounds prepared from amino acids and nucleotide analogues. Fluorescent nucleotide analogues will be used as covalent spectroscopic probes of the nucleotide sites and their interactions with other ligand binding sites. By using affinity labeling reagents which incorporate functional groups at various positions of the ribose or purine ring, it should be possible to map the regions of glutamate dehydrogenase which contribute to the various nucleotide sites. The bovine liver enzyme will be studied as an example of a regulatory enzyme. The yeast enzyme, which has recently been cloned, will be examined as a representative of a non-allosteric enzyme; a collaborative study is planned to evaluate, by site-directed mutagenesis of the gene encoding the yeast enzyme, those amino acid residues which are implicated in function by the technique of affinity labeling. These proposed approaches for studying the structures of glutamate dehydrogenases in solution will complement the X-ray crystallographic studies being conducted on the vertebrate and microbial enzymes.
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