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The conformation of the polypeptide chain in horse liver alcohol dehydrogenase (EC 1.1.1.1), as well as the binding sites for some inhibitor molecules, have been determined from x-ray crystallographic data to a resolution of 2.9 A. The apoenzyme of LADH crystallizes in space-group C2221 with one subunit per asymmetric unit and cell dimensions a = 56.0 A, b = 75.2 A, and c = 181.6 A (10). The crystallographic 2-fold axis relating the two subunits of the apoenzyme molecule is not present in crystals of complexes between apoenzyme and coenzyme, which suggests that the coenzyme may induce a structural asymmetry in the chemically identical subunits. An electron density map to 5-A resolution of the apoenzyme molecule has been described (11).Here we report the structure of this molecule as deduced from an electron-density distribution at a resolution of 2.9 A. Most important is the analysis of the binding sites for some inhibitor molecules, which permits us to identify functional attributes of the enzyme structure; MATERIALS AND METHODSThe enzyme was isolated from fresh horse livers (Akeson, A. & Lundqvist, G., to be published). Preparation of crystals and heavy-atom derivatives suitable for x-ray studies has been described (10). Methods of isomorphous replacement similar to those used for other protein-structure determinations were applied to obtain the electron density map. The crystallographic data were measured at +40 on a computercontrolled Philips-Stoe four-circle diffractometer equipped with a 32 K disc storage. Data were collected to a resolution of 2.9 X from crystals of the native protein, three heavy-metal derivatives [K2Pt(CN)4, KAu(CN)2, and K2Pt(CN)4 + KAu(CN)2], and one inhibitor complex [adenosine diphosphate ribose (ADP-ribose) ]. Intensities within 4.5 A were also measured on two other inhibitor complexes: 8-Br-ADPribose and 1,10-phenanthroline. A skeletal model of the main chain was built with the Kendrew-type models, with an optical comparator (12). CONFORMATION OF THE SUBUNITIn this section we describe briefly the conformation of the polypeptide chain and some details of the subunit interaction and binding of inhibitor molecules as deduced from our electron-density maps. In the next section we will discuss some implications of this structure.The two highest features in our 2.9-A electron-density map were roughly spherical in shape and were interpreted as the two zinc atoms of the subunit. From our 5-A work we already knew the position of one of these zinc atoms and the subunit

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Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys-46, which is a ligand to the active-site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C-labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP-ribose, Pt(CN)i-, I,lO-phenanthroline, Au(CN); and AMP also prevented alkylation with decreasing effectiveness.Crystallographic studies of the alkylated enzyme show that the carboxymethylated sulfur atom of Cys-46 is still liganded to the active-site zinc atom and that theiodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN)i-to the enzyme. It was found that Arg-47 interacts with the phosphate moiety of the nucleotide. Lys-228 and Arg-47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano-ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active-site zinc atom.The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion-binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP-ribose, Pt(CN):-and to a lesser extent Au(CN); and AMP, which therefore prevent the reversible binding of iodoacetate. 1,lO-Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc.Identification of this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active-site zinc region and the general anion-binding site are also presented.