The long term objectives of this investigation are to define and understand the details of the non-redox and redox roles of the pyridine coenzymes in cellular control and energy metabolism. We propose to study, through the use of NAD+ analogues, the non-redox reactions of NAD+ by determining the sterochemistry and studying the mechanism of NAD-glycohydrolases and ADP-ribosyltransferases (diphtheria toxin, cholera toxin and E. coli enterotoxin). We propose to study the factors defining the geometric constraints and binding specificity of dehydrogenases through the synthesis of conformationally rigid NAD derivatives designed to be bisubstrate analogues. The well characterized enzyme, lactate dehydrogenase will be the initial target for these studies. The proposed derivatives represent the first of a series of selective inhibitors targeted for specific dehydrogenases. We propose to investigate the mechanism of dehydrogenases by a multifaceted apporach. We will synthesize compounds designed to model the conformational features of the transition state, conduct 15N NMR experiments to study the transition complexes in the active site and determine the enzymatic properties of NAD analogues designed to be potential mechanism-based inhibitors of both dehydrogenases and NAD-glycohydrolases.
