The enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond of NAD+ and the attendant delivery of the ADPRibosyl moiety to acceptors is central to many diverse biological activities, such as the medically and potentially militarily (biological warfare) important NAD-dependent toxins associated with: cholera, diphtheria, pertussis, and related toxins; the reversible ADPRibosylation-mediated biological regulatory systems; the synthesis of poly(ADPRibose) in response to DNA damage or cellular division; and the synthesis of cyclicADPRibose as part of an apparently independent, yet parallel, calcium-mediated regulatory system. All evidence so far points to these enzymes catalyzing a dissociative cleavage of the nicotinamide-ribosyl bond via an oxocarbenium-like intermediate. How does an enzyme catalyze an SN1 reaction, because by definition such reactions are unimolecular processes? No compelling mechanism has been proposed to account for the ability of these enzymes to catalyze bond scission in the cationic pyridine nucleotides. Yet these enzymes can obviously catalyze the reaction quite effectively. All other glycosidases, nucleosidases, etc. that catalyze dissociative cleavage of glycosyl bonds via an A1 mechanism share the same problem of catalyzing dissociative chemistry. We propose to test a mechanism for the enzyme-catalyzed reaction and those of alternatives. Our mechanism is based on inductive electron donation into the incipient oxocarbenium ion via the hydroxyl of the nicotinamide ribose and concomitant lowering of the entropic barriers to bond scission. We will address fundamental questions regarding the factors that determine the mechanism of nucleophilic substitution reactions. A long term goal of these studies is to use this mechanistic information to help design inhibitors of NAD+ cleaving enzymes that could serve as: chemical antitoxins, probes of nonredox functions of NAD+, and ultimately as models for the design of inhibitors of mechanistically related enzymes that cleave glycosyl bonds, for example, the enzymes that glycosylate the HIV capsid. We have conducted much of this project in collaboration with a group in Strasbourg, France. Now that they are in the process of obtaining detailed structural information on the calf spleen NAD-glycohydrolase we will make use of the Computer Graphics Laboratory in order to understand the mechanism of these enzymes and aid in the design of inhibitors.