The Crystal Structure of Complexes between Horse Liver Alcohol Dehydrogenase and the Coenzyme Analogues 3‐Iodopyridine‐adenine Dinucleotide and Pyridine‐adenine Dinucleotide

Abstract

We have studied the binding of the enzymatically active NAD + analogue, 3-iodopyridine-adenine dinucleotide, and the inactive analogue, pyridine-adenine dinucleotide to the enzyme horse liver alcohol dehydrogenase using X-ray crystallographic methods. These studies were made under such conditions that crystals of the complexes were isomorphous to apocnzyme crystals. Both analogues bind in the same conformation. The binding of the adenosine moiety is very similar to that of ADP-ribose or NADH bound to the enzyme. The conformation and mode of binding of the remaining portions of the analogue molecules is, however, quite different. The pyridine ring is not situated in the active-site pocket as the nicotinamide group in thc isomorphous enzyme -NADH . imidazole complex but lies at the surface of the crevice between the two domains of the subunit, approximately 1.5 nm away from the catalytically active zinc atom. Lys-228 which has been shown to be important for NADH dissociation is in this region of the molecule.A large number of analogues to the coenzyme NAD' have been investigated [l] in order to delineate the role of the various moieties of the dinucleotide in the interaction of this coenzyme with the NAD+-dependent dehydrogenases. We have been particularly interested in the enzymatic effects of substitutions on the nicotinamide ring. As part of these studies, and also for thc use as heavy-atom derivatives in protein Crystallography, we synthesized 3-iOdO and subsequently 3-bromo and 3-chloro derivatives of pyridineadenine dinucleotide which were active in enzymatic hydrogen transfer reactions. Synthesis of these analogues and their kinetic properties for some NAD+-dependent dehydrogenase-catalysed reactions is presented elsewhere [2].The present study describes the binding of the 3-iodo analogue to one of these enzymes, horse liver alcohol dehydrogenase, as studied by X-ray crystallographic methods. Furthermore, the results of that Abbreviations.Pd AD + . pyridine-adenine dinucleot ide ; io3PdAD+, 3-iodopyridine-adenine dinucleotide; Tes, 2-([tris-(hydroxymethyl)methyi]amino)ethane sulfonic acid.

5-Methylnicotinamide-adenine Dinucleotide. Kinetic Investigation with Major and Minor Isoenzymes of Liver Alcohol Dehydrogenase and Structural Determination of Its Binary Complex with Alcohol Dehydrogenase

“…The electron density map is superimposable on the one described for the binding of io3PdADC and PdAD' to liver alcohol dehydrogenase [21]. Part of the electron density is very similar to a part of the electron density of ADP-ribose and was attributed to the adenosine moiety of the molecule [22].…”

“…It is thus not surprising that apoenzyme crystals dissolve by diffusion and binding of such molecules as NADH which induce the conformational transition. ADP-ribose and its 8-bromo derivative, as well as NADH (C. Zeppezauer, personal communication), PdAD' and io3PdAD+ [21], bind to the apo-form of the enzyme in the presence of imidazole [22]. Similarly, the binary complex with the catalytically inactive analogue H,NADH gave orthorhombic crystals, whereas the ternary complex with H,NADH and p-dimethylamino-cinnamaldehyde produced triclinic crystals (Samama and Zeppezauer, unpublished results).…”

“…In the alcohol dehydrogenase-m*NAD+ complex, m'NAD' binds to isoenzyme I11 of horse liver alcohol dehydrogenase in the non-productive mode as was observed for io3PdAD+ and PdAD' which lack the essential amide group [21]. The 5-methylnicotinamide ring is located in an hydrophilic pocket at the surface of the protein molecule, 2 nm away from the nicotinamide moiety bound within the active site in the complex enzyme-NADH-Me,SO [2] (Fig.…”

“…It is quite clear from the studies reported here, that this assumption may not always be valid. This is exemplified by the comparison of the binding of ADP-ribose [22] and PdAD' [21]. The ADP-ribose molecule binds within the active site while PdADf is excluded from this area but still binds to the orthorhombic structure.…”

Binding of Coenzyme and Substrate and Coenzyme Analogues to 6‐Phosphogluconate Dehydrogenase from Sheep Liver

“…imidazole complex. The pyridine ring of io3PdAD' is not in the active-site pocket, but lies at the surface of the crevice between the coenzymebinding domain and the catalytic domain, 1.5 nm from the catalytically active zinc atom [23]. …”