Structure of Liver Alcohol Dehydrogenase at 2.9-Å Resolution

Abstract

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

Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases

“…Long segments of possible but distant homology are found between GPDH and LADH (11) or GDH (12). Similarities in the tertiary structures of LDH (13), soluble malate dehydrogenase (14), LADH (15), and GPDH (16) are also evident. Ancestral connections between different dehydrogenases are therefore likely, although evolutionary relationships are not clear.…”

“…The conserved region in LADH contains the "functional" (35,36) zinc atom (15), which may therefore also occur in YADH. Since there is only one zinc atom per subunit of YADH (17), it would appear that the "structural" (35,36) zinc atom is missing.…”

NAD(P)+ Analogues: Tools for the Investigation of the Active Site of Oestradiol 17beta-Dehydrogenase from Human Placenta

“…The methyl group at C-4 hinders the binding of the analogues to the enzyme. If the binding site of NAD' in oestradiol 17P-dehydrogenase is similar to those of lactate dehydrogenase [ 181, malate dehydrogenase [ 191, and horse liver alcohol dehydrogenase [20], the nicotinamide ring is deeply buried inside the protein, so interaction of the methyl at C-4 with some amino acid side chain may disfavour the binding. Partial binding of these analogues by the adenine part is also weak.…”

The Conformation of Adenosine Diphosphoribose and 8-Bromoadenosine Diphosphoribose when Bound to Liver Alcohol Dehydrogenase

“…The crystal structure determination of horse liver alcohol dehydrogenase has been carried out on crystals of the apoenzyme [1,2]. Complexes of liver alcohol dehydrogenase with coenzyme invariably crystallize in different crystal modifications compared to the coenzyme-free crystals.…”