Eklund H, Samama J P, Jones T A
Biochemistry. 1984 Dec 4;23(25):5982-96. doi: 10.1021/bi00320a014.
The binding of NAD to liver alcohol dehydrogenase has been studied in four 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 has been refined at 2.9-A 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 with hydrophobic interactions between two isoleucine side chains. Both ribose rings have 2E(C2'-endo) puckering, and each ribose makes three hydrogen bonds to the enzyme. The pyrophosphate bridge has hydrogen bonds to the side chains of arginine-47 and -369 and to main chain nitrogen atoms from the amino ends of two alpha-helices. The nicotinamide ring is in van der Waals contact with the active-site zinc atom and with the sulfur atoms of its cysteine ligands. The carboxamide group is about 30 degrees out of the plane of the nicotinamide ring and hydrogen 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 three 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 one entrance to the active site. As a comparison, ADP-ribose binding has been studied in the open form of the enzyme. The adenosine moiety binds in a similar way as NAD, while the rest of the molecule has different interactions.
通过晶体学方法，在四种不同的三元复合物中研究了NAD与肝脏乙醇脱氢酶的结合。这些复合物以三斜晶系晶体形式同晶型结晶，其不对称单元中包含酶的整个二聚体。这种形式的酶已在2.9埃分辨率下精修至晶体学R因子为0.22。在这些复合物中，NAD的结合方式基本相同。结合位点位于辅酶结合结构域的中心部分。腺嘌呤环通过两个异亮氨酸侧链之间的疏水相互作用结合。两个核糖环均具有2E（C2'-内型）褶皱，并且每个核糖与酶形成三个氢键。焦磷酸桥与精氨酸-47和-369的侧链以及来自两个α-螺旋氨基末端的主链氮原子形成氢键。烟酰胺环与活性位点锌原子及其半胱氨酸配体的硫原子处于范德华接触。羧酰胺基团与烟酰胺环平面大约成30度角，并与残基292、317和319的主链原子形成氢键。NAD分子的整体构象与其他脱氢酶观察到的相似，但在细节上有所不同。在辅酶存在下，酶经历从开放形式到封闭形式的大的构象变化。这种构象变化有三个主要作用：与酶的基团产生有利的结合相互作用，包围辅酶并通过减少可及表面积获得辅酶的结合能，以及封闭活性位点的一个入口。作为比较，已在酶的开放形式中研究了ADP-核糖的结合。腺苷部分的结合方式与NAD相似，而分子的其余部分具有不同的相互作用。
