Structural and functional zinc in horse liver alcohol dehydrogenase.

Abstract

The location of amino acid side chains-whether at the surface or in the interior of protein molecules-and their immediate chemical environment are thought to be critical to their chemical reactivity toward modifying agents. Certain catalytically essential amino acid residues of enzymes have proven particularly reactive chemically. Similarly, metal atoms of metalloproteins can exhibit differential reactivity related to their location and roles. This is exemplified by recent studies of horse liver alcohol dehydrogenase (LADH).'Materials and Mfethods.-Crystalline horse LADH isolated by a modification of the Dalziel procedure2 and homogeneous by ultracentrifugation was obtained from Boehringer M\Iannheim Corporation. By chromatography on carboxymethyl cellulose, about 95 per cent of the protein and enzymatic activity eluted together in a single symmetrical peak in a position corresponding to the major component described by Dalziel3 and contained 3.4-3.6 gm at. of Zn/mol wt 80,000. However, electrophoresis on cellulose acetate and starch gel at pH 8.5 revealed the presence of five bands, as demonstrated by staining techniques both for protein and enzymatic activity. About 75 per cent of the protein and the enzymatic activity were associated with the third and 25 per cent with the fourth cathodial bands, in accord with the findings of Pietruszko et al.4 5 Urea was recrystallized from ethanol and aqueous solutions prepared immediately before use. The control of metal contamination and the determinations of enzymatic and isotopic activities, protein concentration, and Zn content have been described.6 A Cary model 60 spectropolarimeter was employed for the optical rotatory dispersion studies. Velocity sedimentation and sedimentation equilibrium were performed in a Spinco model E ultracentrifuge.The apparent partial specific volume7 and density were determined pycnometrically.Results and Discussion.-Zinc content: LADH contains two active enzymatic centers, each binding one molecule of coenzyme.8 The Zn content of early preparations ranged from 1.7 to 2.8, with a mean of 2.1 gm at. of Zn based on a mol wt of 73,000.9' 10 Both by spectral' and by kinetic12 criteria, two moles of 1,10-phenanthroline (OP) competed with coenzyme and were thought to bind at or near the Zn1 atoms of the two active centers. However, the enzyme then employed contained substantial admixtures of impurities, largely eliminated by subsequent modifications of the isolation procedure as evidenced by specific enzymatic activity, sulfhydryl content, and chromatographic criteria.2 3 The enzyme prepared in this manner had a mol wt of 84,000.13 The amount of Zn found earlier9' 10 would be equivalent to 2-3.2 gm at. of Zn per mol wt 84,000. Carboxymethylation with C14-iodoacetate inactivates LADH while labeling the cysteinyl residues of two identical peptides,'4' 15 suggesting two active sites and two identical subunits per mole.The Zn content of more highly purified preparations has been found to be 4 gm at.1434

Differences between Alcohol Dehydeogenases

“…This could explain previously noticed inconsistencies in structural comparisons [13, 141 and the complete loss [42] or apparently altered binding [43] of one zinc atom per subunit in the active yeast enzyme. This second zinc atom has no known function [14,36], although earlier considered to stabilize the tertiary structure [44,45] and is probably not conserved in evolution of alcohol dehydrogenases as shown from analyses of the corresponding Drosophila protein [32]. It therefore seems possible that the deleted region in the yeast enzyme may affect surface properties, binding of the second zinc atom and subunit interactions, which is compatible with known differences of the enzymes in quaternary structure and zinc content.…”

Effect of zinc status of rats on the synthesis and degradation of copper-induced metallothioneins

“…However, the binding of various compounds to proteins can alter their rate of catabolism (Goldberg & Dice, 1974), as is illustrated by the retardation in the degradation of ferritin caused by the binding of Fe3+ to the protein (Drysdale & Munro, 1966). The stabilkzing effect of Zn2+ on copper-thionein could be consistent with one of the functional roles of Zn2+ in maintaining the structural integrity of certain zincenzymes (Drum et al, 1967) and possibly in preventing oxidation of thiol groups (Chvapil, 1973). It is interesting that the loss of Zn2+ which occurred during the purification of renal copper-thioneins from sheep was associated with considerable molecularweight changes in the proteins, consistent with the occurrence of oxidation and polymerization reactions between copper and exposed thiol groups (Bremner & Young, 1977).…”

Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases

“…Since there is only one zinc atom per subunit of YADH (17), it would appear that the "structural" (35,36) zinc atom is missing. Its function in the dimeric LADH may, presumably, therefore be replaced by the tetrameric structure of YADH.…”

“…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.…”