Is NAD+ a substrate or a product?

Is NAD+ a substrate or a product?

NAD+ is both. It is a substrate when an enzyme uses it up, and a product when an enzyme makes it. A substrate is the molecule that goes into a reaction. A product is the molecule that comes out. Covarrubias et al. describe cells that keep NAD+ in balance through making it, using it up and recycling it (2021). So the label depends on which reaction you look at.

The enzyme records on this page show the pattern. Lactate dehydrogenase, sirtuins, PARPs and CD38 take NAD+ in. NMNAT enzymes, NAD synthase and complex I give NAD+ out. The sections below take them in turn.

When is NAD+ a substrate in redox reactions?

NAD+ is a substrate when a redox enzyme uses it to take up electrons. Redox means a reaction that moves electrons. ExPASy ENZYME lists L-lactate dehydrogenase (EC 1.1.1.27) with the reaction (S)-lactate + NAD+ = pyruvate + NADH + H+. NAD+ goes in and NADH comes out. NADH is the same coenzyme after it accepts electrons. Rhea holds the same equation as reaction 23444.

ChEBI lists NAD+ as C21H28N7O14P2 with charge +1 and NADH as C21H29N7O14P2 with charge 0 (ChEBI CHEBI:15846, ChEBI CHEBI:16908). The extra hydrogen on NADH fits the electrons NAD+ took up. The equation uses an equals sign. A reaction written that way can be read in both directions. Read from right to left, NAD+ is a product.

Which enzymes use NAD+ as a substrate and break it down?

Sirtuins, PARPs and CD38 use NAD+ as a substrate and break it down. Covarrubias et al. group these as the NAD+-consuming enzymes: the NAD+ glycohydrolases (CD38, CD157 and SARM1), the sirtuins and the PARPs (2021). Each one splits NAD+ and releases nicotinamide (NAM), a small part of the molecule. ExPASy ENZYME gives each reaction an EC number, which is a standard code for an enzyme reaction.

Sirtuins are listed as EC 2.3.1.286. The reaction is N(6)-acetyl-L-lysyl-[protein] + NAD+ + H2O = 2'-O-acetyl-ADP-D-ribose + nicotinamide + L-lysyl-[protein] (Rhea 43636). PARPs are listed as EC 2.4.2.30. In that reaction, an ADP-ribose group from NAD+ goes onto an acceptor and nicotinamide is released. CD38 is listed under EC 3.2.2.6, with the reaction NAD+ + H2O = ADP-D-ribose + nicotinamide + H+ (Rhea 16301).

In all three, NAD+ is a substrate and nicotinamide is a product. This is a description of what the enzymes do to a molecule. It is not a claim about what happens in a person.

Which enzymes make NAD+ as a product?

NMNAT enzymes and NAD synthase make NAD+ as a product. ExPASy ENZYME lists nicotinamide-nucleotide adenylyltransferase (EC 2.7.7.1) with the reaction beta-nicotinamide D-ribonucleotide + ATP + H+ = diphosphate + NAD+. Covarrubias et al. name three human forms, NMNAT1, NMNAT2 and NMNAT3, that turn nicotinamide mononucleotide (NMN) into NAD+ (2021). Rhea holds the NMNAT reaction as 21360.

NAD synthase is a second maker. The glutamine-using form is EC 6.3.5.1: deamido-NAD+ + L-glutamine + ATP + H2O = L-glutamate + AMP + diphosphate + NAD+ + H+ (Rhea 24384). A form that uses ammonia is EC 6.3.1.5 (Rhea 21188).

Covarrubias et al. say that cells recycle the nicotinamide that sirtuins, PARPs and CD38 release. The chain goes from nicotinamide to NMN and then to NAD+ (2021). So a product of one enzyme set, nicotinamide, becomes the starting material for the next. The full pathways are on the how NAD+ is made page.

Is NAD+ a product when NADH is oxidised?

Yes. NAD+ is a product when an enzyme oxidises NADH, which means it removes the electrons NADH carries. ExPASy ENZYME lists NADH:ubiquinone reductase (EC 7.1.1.2), also called complex I. The reaction is a ubiquinone + NADH + 5 H+(in) = a ubiquinol + NAD+ + 4 H+(out). NADH is the substrate here and NAD+ is a product (Rhea 29091).

This reaction is the lactate dehydrogenase pattern in reverse. In the first, NAD+ takes electrons and becomes NADH. Here, NADH gives electrons away and becomes NAD+. The same coenzyme moves between two forms. Each form is a substrate for some enzymes and a product of others.

Is NAD+ a substrate, a coenzyme or a cofactor?

NAD+ is a coenzyme, and in many reactions it is also a substrate. ChEBI defines a coenzyme as a small, non-protein organic compound that acts as a dissociable acceptor or donor of chemical groups or electrons. Dissociable means it can come off the enzyme. Covarrubias et al. call NAD+ a coenzyme for redox reactions and an essential cofactor for sirtuins, CD38 and PARPs (2021). The three words describe how an enzyme uses the molecule.

In the lactate dehydrogenase record, NAD+ is a reactant that is changed into NADH. That is why the record lists it as a substrate. In the sirtuin, PARP and CD38 records, the enzyme splits NAD+ apart. A cofactor is a helper an enzyme needs. A substrate is what the enzyme works on. NAD+ fills both roles.