The role of NAD in the urocanase reaction is under study. The overall reaction involves the hydration of urocanic acid to 4-imidazolone-5-propionate, but the recent discovery that urocanase of Pseudomonas putida contains a tightly bound NAD suggests that the reaction proceeds through an internal oxidation-reduction. Isotopic studies of intramolecular hydrogen transfers are anticipated in order to elucidate the manner in which NAD acts. Additional studies are planned on two threonine dehydratases. The first, from P. putida, has been shown to be essential for formation of histidine degrading enzymes in this organism. We propose to investigate how these processes are linked, by analysis of mutants lacking or altered in threonine dehydratase, so as to determine whether this connection is indirectly mediated or whether threonine dehydratase itself is necessary for hut operon mRNA formation. The second threonine dehydratase to be studied is the ADP-activated enzyme from Clostridium tetanomorphum. We are actively studying the ligand-induced oligomerization of the enzyme, especially that promoted by L-threonine. In addition, we are mapping the binding sites for ADP and threonine in order to obtain information as to their physical location relative to one another.