Structure of Horse Liver Alcohol Dehydrogenase. II. Heavy-atom Derivatives of Type A Crystals.

Söderberg, Bengt-Olof; Zeppezauer, E.; Boive, T.; Nordström, B.; Brändén, Carl‐Ivar

doi:10.3891/acta.chem.scand.24-3567

Cited by 13 publications

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“…The idea of negative cooperativity governing the simultaneous binding of Pt(CN),,-and Au(CN),-is supported by the crystallographic data of Soderberg et al [12]. In the ternary enzyme * Pt(CN),,-Au-(CN)2-complex, the occupancy of both platinum and gold is reduced to about half the values found in the respective binary enzyme anion complexes.…”

Section: Discussionmentioning

confidence: 88%

“…This observation is of interest since Pt(CN),,-and Au(CN),-have been used for the preparation of crystalline heavy-atom derivatives of the enzyme [12] and their binding position can be established by crystallographic techniques. While a detailed spectroscopic study of these crystalline protein derivatives is under way [13], we have been investigating the mode of binding of Pt(CN),2-and Au(CN),-in solution, as indicated by their effect on the kinetic properties of the enzyme.…”

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confidence: 99%

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Inhibition of Horse‐Liver Alcohol Dehydrogenase by Pt(CN)₄^2– and Au(CN)₂^–

Gunnarsson¹,

Pettersson²,

Zeppezauer

1974

European Journal of Biochemistry

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Single and multiple-inhibition experiments provide evidence that the complex anion Pt,(CN),2-is bound a t a single site per subunit of liver alcohol dehydrogenase in strict competition with coenzyme, ADP-ribose, AMP and (less than 100 mM) chloride ion, but independently of 1,IOphenanthroline. Binding of adenosine decreases the stability constant for the enzyme . Pt(CN),2-complex from 16 mM-l to 4 mM-l.Au(CN),-is bound at two sites per subunit of the enzyme. Both interactions are competitive with coenzyme, ADP-ribose and AMP, but independent of 1 ,lo-phenanthroline. Only one site is available for interaction with Au(CN),-in the binary enzyme complexes formed with Pt(CN),2-, chloride ion, or adenosine. Estimation of the corresponding binding constants for Au(CN),-gave evidence for cooperativity between the two Au(CN),--binding sites.These results strongly indicate that Pt(CN),2-is bound a t a site within the protein region occupied by the phosphate groups of the coenzyme, and it is suggested that arginine-47 in the enzymatic amino-acid sequence is part of a common binding site for negative charges on the coenzyme and coenzyme competitive anions such as Pt(CN),Z-, Au(CN),-, chloride and iodoacetate. The second binding site for Au(CN),-appears to be situated within the protein region occupied by the adenosine part of the coenzyme.

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Section: Discussionmentioning

confidence: 88%

mentioning

confidence: 99%

Inhibition of Horse‐Liver Alcohol Dehydrogenase by Pt(CN)₄^2– and Au(CN)₂^–

Gunnarsson¹,

Pettersson²,

Zeppezauer

1974

European Journal of Biochemistry

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“…& 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.…”

Section: Methodsmentioning

confidence: 99%

Structure of Liver Alcohol Dehydrogenase at 2.9-Å Resolution

Brändén¹,

Eklund²,

Nordström³

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

175

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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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“…[Au(CN) 2 - ], used as a crystallographic heavy-atom probe, binds intact to haloalkane dehydrogenase (HD), human carbonic anhydrase I, horse liver alcohol dehydrogenase, and human liver interleukin-1 receptor antagonist protein . In HD, iodide can bind at the same site .…”

Section: Resultsmentioning

confidence: 99%

¹⁹⁷Au Mössbauer Characterization of the Noncovalent Adducts Formed between Serum Albumin and Dicyanoaurate(I), a Gold-Drug Metabolite

1999

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Chrysotherapy is an important treatment for rheumatoid arthritis. 1,2 Metabolic transformations of the first-and secondgeneration gold drugs (Myochrysine, Solganol, and auranofin) begin with the displacement of their thiolate ligands via ligand exchange reactions with serum proteins and may culminate at inflammatory sites where dicyanoaurate(I) is formed. 3 It is a common metabolite of all three gold drugs, found in the serum and urine of patients, 4 and must be transported from the inflamed sites to the kidneys. Serum albumin carries up to 95% of the serum gold, and may be a transport agent for [Au(CN) 2 -].The dicyanoaurate(I) ion is linear with d(Au-C) ) 197.1(1) pm, d(CN) ) 146(1) pm, and ∠C-Au-C ) 179(2)°for three independent ions in Tl[Au(CN) 2 ] 5 and has an extremely large binding constant, log β 2 ) 36.6. 6 13 C NMR studies of albumindicyanoaurate(I) complexes, their equilibrium binding constants (K 1 ) 5.5 × 10 4 and K 2 ) 5.5 × 10 3 ), and the similarity of reactions of native and sulfhydryl-modified albumin all provide indirect evidence that multiple, intact dicyanoaurate(I) ions bind to albumin, 7,8 unlike gold drugs which undergo ligand exchange reactions preferentially or exclusively at cysteine 34. [9][10][11] Mo ¨ssbauer spectroscopy using the 77 keV resonance of 197 Au is a suitable technique for characterizing gold complexes [12][13][14][15][16][17] and is applicable to noncrystalline materials. It provides a method to distinguish among (i) adduct formation by intact dicyanoaurate-

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Structure of Horse Liver Alcohol Dehydrogenase. II. Heavy-atom Derivatives of Type A Crystals.

Cited by 13 publications

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Inhibition of Horse‐Liver Alcohol Dehydrogenase by Pt(CN)₄^2– and Au(CN)₂^–

Inhibition of Horse‐Liver Alcohol Dehydrogenase by Pt(CN)₄^2– and Au(CN)₂^–

Structure of Liver Alcohol Dehydrogenase at 2.9-Å Resolution

¹⁹⁷Au Mössbauer Characterization of the Noncovalent Adducts Formed between Serum Albumin and Dicyanoaurate(I), a Gold-Drug Metabolite

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Structure of Horse Liver Alcohol Dehydrogenase. II. Heavy-atom Derivatives of Type A Crystals.

Cited by 13 publications

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Inhibition of Horse‐Liver Alcohol Dehydrogenase by Pt(CN)42– and Au(CN)2–

Inhibition of Horse‐Liver Alcohol Dehydrogenase by Pt(CN)42– and Au(CN)2–

Structure of Liver Alcohol Dehydrogenase at 2.9-Å Resolution

197Au Mössbauer Characterization of the Noncovalent Adducts Formed between Serum Albumin and Dicyanoaurate(I), a Gold-Drug Metabolite

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Inhibition of Horse‐Liver Alcohol Dehydrogenase by Pt(CN)₄^2– and Au(CN)₂^–

Inhibition of Horse‐Liver Alcohol Dehydrogenase by Pt(CN)₄^2– and Au(CN)₂^–

¹⁹⁷Au Mössbauer Characterization of the Noncovalent Adducts Formed between Serum Albumin and Dicyanoaurate(I), a Gold-Drug Metabolite