This renewal proposal will continue ongoing studies on the mechanism of Salmonella histidinol dehydrogenase (HDH), a 4- electron NAD-linked enzyme. The focus is on the use of primary isotope effects to obtain rates for the two reaction steps, the chemistry of the steps, and the pK values and nature of the enzyme residues involved. A high priority is assigned to completing the stereochemistry of the dehydrogenation reactions. Although important binding interactions of histidinol with HDH at the imidozole ring and amino regions have been identified, the relative positions of these two groups in space are not known. Rigid histamine receptor agonists will be used to determine this topography. In order to identify the active site residues involved in binding or catalysis, site directed modification and mutagenesis will be used. Logical modifiers are being synthesized. In addition to the inherent enzyme mechanistic interest in HDH, the system is of broad importance in biology. The enzyme is coded for by the hisD gene of the Salmonella histidine operon, a particularly well studied genetic system. The known mutants in the system form the basis for the well known Ames test.
