CRYSTALLOGRAPHIC INVESTIGATIONS OF NICOTINAMIDE ADENINE-DINUCLEOTIDE BINDING TO HORSE LIVER ALCOHOL-DEHYDROGENASE

The binding of NAD to liver alcohol dehydrogenase was studied in 4 different ternary complexes by using crystallographic methods. These complexes crystallize isomorphously in a triclinic crystal form which contains the whole dimer of the enzyme in the asymmetric unit. This form of the enzyme was refined at 2.9-.ANG. resolution to a crystallographic R factor of 0.22. NAD binds in essentially the same way in these complexes. The binding site is located at the central part of the coenzyme binding domain. The adenine ring binds which hydrophobic interactions between 2 isoleucine side chains. Both ribose rings have 2E(C2''-endo) puckering, and each ribose makes 3 H-bonds to the enzyme. The pyrophosphate bridge has H-bonds to the side chains of arginine-47 and -369 and to main chain N atoms from the amino ends of 2 .alpha.-helices. The nicotinamide ring is in van der Waals contact with the active site Zn atom and with the S atoms of its cysteine ligands. The carboxamide group is about 30% out of the plane of the nicotinamide ring and H-bonds to main chain atoms of residues 292, 317 and 319. The overall conformation of the NAD molecule is similar to that observed for other dehydrogenases, but differs in details. In the presence of the coenzyme, the enzyme undergoes a large conformational change from an open to a closed form. This conformational change has 3 major effects: to create favorable binding interactions with groups of the enzyme, to enclose the coenzyme and gain binding energy for the coenzyme by reducing the accessible surface area, and to close off 1 entrance to the active site. As a comparison, ADP-ribose binding was studied in the open form of the enyzme. The adenosine moiety binds in a similar way as NAD, while the rest of the molecule has different interactions.