Principles of Protein Structure - H. Schulz 1982

Protein-ligand interactions
Nucleotide-binding sites
NAD-binding domains of dehydrogenases

The structures of four NAD-dependent dehydrogenases have been determined: Lactate dehydrogenase (LDH) [232], s-malate dehydrogenase (MDH) [233], Liver Alcohol dehydrogenase (ADH), and D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [230, 231]. These Enzymes catalyze The transfer of a hydride ion from their respective substrates to the C4 atom of the nicotinamide ring of NAD. Each enzyme subunit contains a single NAD-binding domain.

All NAD-binding domains contain parallel ß-structures with identical topology. The domains are homologous [91] due to the characteristic conserved topology of their ß-pleated sheets (Section 9-6). Specifically, the arrangement of ß-structures within the NAD-binding region is strictly conserved, as shown schematically in Fig. 7.7. This conserved spatial arrangement has persisted despite numerous Amino Acid Substitutions: there is no notable Amino Acid Sequence Homology between any two of these domains.

NAD binds in an extended conformation. The binding of NAD to any of the four dehydrogenases in this characteristic conformation reflects the structural similarity of their NAD-binding domains. The adenine and nicotinamide rings are separated by 14 Å, and their planes are nearly perpendicular to each other. It is useful to consider the adenosine and nicotinamide moieties of NAD separately. One reason for this is that the NAD-binding domain itself may have evolved through the duplication of a smaller structural unit, the Rossmann fold (Section 9.4). As shown schematically in Fig. 7.7, each mononucleotide fragment of NAD binds to a single Rossmann fold.

The adenine-binding pocket is not highly specific. The adenosine moiety of NAD binds in a pocket that lacks strict Specificity for this Ligand, accommodating aromatic residues in general. The only invariant residue in the adenosine-binding sites of all four dehydrogenases is the last residue of the first ß-pleated sheet in the Rossmann fold (Fig. 5.12, b). This residue is Glycine; any larger side chain would sterically hinder the binding of the ribosyl moiety. Also conserved is the terminal residue of the second ß-sheet, which is an aspartic acid that forms a Hydrogen bond with the O-2' atom of the ribose—an interaction crucial for the ability of these enzymes to discriminate between NAD and NADP.

In LDH, ADH, and MDH, the hydride ion is transferred to the A-side of the nicotinamide ring, whereas in GAPDH it is transferred to the B-side. The nicotinamide moiety of NAD is invariably bound in a cavity that is hydrophobic on one side and hydrophilic on the other, interacting with substrates such as lactate, ethanol, malate, or glyceraldehyde-3-phosphate. The hydride ion is transferred stereospecifically to the nicotinamide ring. In LDH, ADH, and MDH, the so-called A-side [85] of the nicotinamide ring interacts with substrates and accepts the hydride ion, whereas in GAPDH the nicotinamide ring is rotated by 180° about the glycosidic bond linking the ribose and nicotinamide, positioning the B-side toward the substrate to accept the hydride ion. This relative orientation is evidently dictated by the spatial constraints of the NAD-binding site.