The overall objectives of this research are to elucidate mechanisms for catalysis and regulation of glutamine amidotransferases. Many glutamine amidotransferases exhibit the capacity to utilize glutamine or NH3 for biosynthetic amination reactions. In some enzymes glutamine and NH3 bind to distinct sites and the amide of glutamine is transferred to the NH3 site prior to amination of the second substrate. Experiments will be conducted to characterize the putative domain for glutamine binding and amide transfer. The cysteine residue that forms the covalent glutaminyl and glutamyl intermediates will be alkylated with a radioactive glutamine affinity analog. The amino acid sequence of the active site region of several amidotransferases will be determined in order to see if similarities in catalytic properties are due to similarities in primary structure. Immunological methods will also be used to search for structural similarity in a glutamine binding domain for amidotransferases. The main enzymes to be investigated are anthranilate synthetase, GMP synthetase, glutamate synthetase and amidophosphoribosyltransferase. In anthranilate synthetase, the glutamine and NH3 sites are on dissimilar protein chains. In GMP synthetase and amidophosphoribosyl transferase these sites are independent but are on the same protein chain. Glutamate synthetase is more complex and therefore a number of experiments are planned to study the mechanism of catalysis.