The main role of NAD in metabolism is the transfer of electrons from one molecule to another. Reactions of this type are catalyzed by a large group of enzymes called oxidoreductases. The correct names for these enzymes contain the names of both their substrates: for example NADH-ubiquinone oxidoreductase catalyzes the oxidation of NADH by coenzyme Q. However, these enzymes are also referred to as dehydrogenases or reductases, with NADH-ubiquinone oxidoreductase commonly being called NADH dehydrogenase or sometimes coenzyme Q reductase.
There are many different superfamilies of enzymes that bind NAD / NADH. One of the most common superfamilies includes a structural motif known as the Rossmann fold. The motif is named after Michael Rossmann, who was the first scientist to notice how common this structure is within nucleotide-binding proteins.
An example of a NAD-binding bacterial enzyme involved in amino acid metabolism that does not have the Rossmann fold is found in Pseudomonas syringae pv. tomato (; ).
When bound in the active site of an oxidoreductase, the nicotinamide ring of the coenzyme is positioned so that it can accept a hydride from the other substrate. Depending on the enzyme, the hydride donor is positioned either "above" or "below" the plane of the planar C4 carbon, as defined in the figure. Class A oxidoreductases transfer the atom from above; class B enzymes transfer it from below. Since the C4 carbon that accepts the hydrogen is prochiral, this can be exploited in enzyme kinetics to give information about the enzyme's mechanism. This is done by mixing an enzyme with a substrate that has deuterium atoms substituted for the hydrogens, so the enzyme will reduce NAD by transferring deuterium rather than hydrogen. In this case, an enzyme can produce one of two stereoisomers of NADH.
Despite the similarity in how proteins bind the two coenzymes, enzymes almost always show a high level of specificity for either NAD or NADP. This specificity reflects the distinct metabolic roles of the respective coenzymes, and is the result of distinct sets of amino acid residues in the two types of coenzyme-binding pocket. For instance, in the active site of NADP-dependent enzymes, an ionic bond is formed between a basic amino acid side-chain and the acidic phosphate group of NADP. On the converse, in NAD-dependent enzymes the charge in this pocket is reversed, preventing NADP from binding. However, there are a few exceptions to this general rule, and enzymes such as aldose reductase, glucose-6-phosphate dehydrogenase, and methylenetetrahydrofolate reductase can use both coenzymes in some species.