We plan to launch a comprehensive effort toward understanding, at a fundamental level, the chemistry of the pyridine nucleotides. We then propose to apply this knowledge to understand the mechanisms of pyridine coenzyme-dependent enzymes. Our tools will be traditional physical organic chemistry to study the chemical properties of pyridine nucleotides, bioorganic chemistry to design and synthesize a wide range of pyridine nucleotide analogues to test our proposed mechanisms and an enzymological approach to evaluate their properties with both dehydrogenases and hydrolytic enzymes. We will investigate the mechanisms of the chemical hydrolysis of the nicotinamide glycosyllinkage in new NAD+ analogues designed to address specific mechanistic questions regarding acid and base catalysis of glycosyl hydrolysis. We will use this information to probe the mechanism of the enzymatic hydrolysis of the nicotinamide glycosyl bond, focusing on the NAD-glycohydrolases and ADP-ribosyltransferases, including the NAD+-dependent cholera and diphtheria toxin. We will also study the chemical and enzymatic properties of reduced forms of acyclic NAD+ analogues as a potential new class of substrate-dependent, active site-directed inactivators of dehydrogenases. Finally we will explore the stereochemical constraints of the coenzyme-dehydrogenase interaction with the nicotinamide moiety in order to determine the interactions that are responsible for the fidelity of stereospecific hydride transfer. The investigations will allow us to achieve a deeper understanding of the functioning of this vitally important class of coenzymes and the mechanism by which enzymes act upon them.
