Polymorphism of horse liver alcohol dehydrogenase

The properties of the most cathodal component of horse liver alcohol dehydrogenase (isozyme SS) have been found to vary. The variability is dependent on the livers from which the enzyme is isolated rather than on the purification procedure. Two distinct preparations, differing in catalytic properties, have been obtained and named S-type and A-type preparations. The preparations can be distinguished from each other by the ratio of activity with acetaldehyde to activity with the steroidal ketone 5_β-dihydrotestosterone. This ratio is about one for the S-type and twenty for the A-type preparations.     

Reactive lysine residues in horse liver alcohol dehydrogenase

Horse liver alcohol dehydrogenase was modified under various conditions with ¹⁴C-labelled formaldehyde in the presence of sodium borohydride. Changes in the enzymatic activity were correlated with incorporated label and modified residues were characterized. It is shown that most of the lysine residues react and that many are affected by the binding of coenzymes and inhibitors to the protein. Reactive residues are reported and possible structural and functional interpretations given.     

Cobalt exchange in horse liver alcohol dehydrogenase.

The preparation of metal hybrid species of horse liver alcohol dehydrogenase is made possible by the development of carefully delineated systems of metal in equilibrium metal exchange employing equilibrium dialysis. The conditions which are optimal for the site-specific replacement of the catalytic and/or noncatalytic zinc atoms of the native enzyme by cobalt are not identical with those which are utilized for substitution with 65Zn. Thus, while certain 65Zn hybrids can be prepared by exploiting the differential effects of buffer anions, the cobalt hybrids are generated by critical adjustments in the pH of the dialysate. Factors which may determine the mechanism of metal replacement reactions include acid-assisted, ligand-assisted, and metal-assisted dechelation, steric restriction, and ligand denticity as well as physicochemical properties of the enzyme itself. The spectral characteristics of the catalytic and noncatalytic cobalt atoms reflect both the geometry of the coordination complexes and the nature of the ligands and serve as sensitive probes of these loci in the enzyme.     

Triplet-singlet energy transfer in the complex of auramine O with horse liver alcohol dehydrogenase

Triplet-singlet energy transfer has been studied in the complex formed between auramine O (AO) and horse liver alcohol dehydrogenase with optically detected magnetic resonance (ODMR) spectroscopy. The results show that Trp-15 and Tyr residues transfer triplet energy mainly by a trivial process, whereas Trp-314 transfers triplet energy by a F?rster process with two observed lifetimes at 77 K of 170 and 50 ms. The different F?rster energy-transfer lifetimes are ascribed either to quenching of the two Trp-314 residues of the dimer by a single asymmetrically bound AO or to two distinct conformations of the enzyme-dye complex with differing separations and/or orientations of donor and acceptor. Individual spin sublevel transfer rate constants are reported for the major decay component with the 170-ms Trp triplet-state lifetime; these are found to be highly selective with kxtr much greater than kytr and kztr.     

Self-association of lyophilized horse liver alcohol dehydrogenase.

The molecular weights of lyophilized and non-lyophilized horse liver alcohol dehydrogenase have been compared by quasi-elastic light scattering, and ultracentrifugation. Whereas the non-lyophilized enzyme has the expected molecular weight of 78 000, the lyophilized enz)me has an initial molecular weight of about 10(6) which increases with time by an endothermic process. This result shows that any physical measurement using lyophilized liver alcohol dehydrogenase to investigate the enzyme mechanism, which relies upon the molecular size, will be invalid.     

Ternary complexes of liver alcohol dehydrogenase

Liver alcohol dehydrogenase (LADH; E.C. 1.1.1.1) provides an excellent system for probing the role of binding interactions with NAD(+) and alcohols as well as with NADH and the corresponding aldehydes. The enzyme catalyzes the transfer of hydride ion from an alcohol substrate to the NAD(+) cofactor, yielding the corresponding aldehyde and the reduced cofactor, NADH. The enzyme is also an excellent catalyst for the reverse reaction. X-ray crystallography has shown that the NAD(+) binds in an extended conformation with a distance of 15 A between the buried reacting carbon of the nicotinamide ring and the adenine ring near the surface of the horse liver enzyme. A major criticism of X-ray crystallographic studies of enzymes is that they do not provide dynamic information. Such data provide time-averaged and space-averaged models. Significantly, entries in the protein data bank contain both coordinates as well as temperature factors. However, enzyme function involves both dynamics and motion. The motions can be as large as a domain closure such as observed with liver alcohol dehydrogenase or as small as the vibrations of certain atoms in the active site where reactions take place. Ternary complexes produced during the reaction of the enzyme binary entity, E-NAD(+), with retinol (vitamin A alcohol) lead to retinal (vitamin A aldehyde) release and the enzyme binary entity E-NADH. Retinal is further metabolized via the E-NAD(+)-retinal ternary complex to retinoic acid (vitamin A acid). To unravel the mechanistic aspects of these transformations, the kinetics and energetics of interconversion between various ternary complexes are characterized. Proton transfers along hydrogen bond bridges and NADH hydride transfers along hydrophobic entities are considered in some detail. Secondary kinetic isotope effects with retinol are not particularly large with the wild-type form of alcohol dehydrogenase from horse liver. We analyze alcohol dehydrogenase catalysis through a re-examination of the reaction coordinates. The ground states of the binary and ternary complexes are shown to be related to the corresponding transition states through topology and free energy acting along the reaction path.     

Ternary complexes of liver alcohol dehydrogenase

Liver alcohol dehydrogenase (LADH; E.C. 1.1.1.1) provides an excellent system for probing the role of binding interactions with NAD⁺ and alcohols as well as with NADH and the corresponding aldehydes. The enzyme catalyzes the transfer of hydride ion from an alcohol substrate to the NAD⁺ cofactor, yielding the corresponding aldehyde and the reduced cofactor, NADH. The enzyme is also an excellent catalyst for the reverse reaction. X-ray crystallography has shown that the NAD⁺ binds in an extended conformation with a distance of 15 Å between the buried reacting carbon of the nicotinamide ring and the adenine ring near the surface of the horse liver enzyme. A major criticism of X-ray crystallographic studies of enzymes is that they do not provide dynamic information. Such data provide time-averaged and space-averaged models. Significantly, entries in the protein data bank contain both coordinates as well as temperature factors. However, enzyme function involves both dynamics and motion. The motions can be as large as a domain closure such as observed with liver alcohol dehydrogenase or as small as the vibrations of certain atoms in the active site where reactions take place. Ternary complexes produced during the reaction of the enzyme binary entity, E-NAD⁺, with retinol (vitamin A alcohol) lead to retinal (vitamin A aldehyde) release and the enzyme binary entity E-NADH. Retinal is further metabolized via the E-NAD⁺-retinal ternary complex to retinoic acid (vitamin A acid). To unravel the mechanistic aspects of these transformations, the kinetics and energetics of interconversion between various ternary complexes are characterized. Proton transfers along hydrogen bond bridges and NADH hydride transfers along hydrophobic entities are considered in some detail. Secondary kinetic isotope effects with retinol are not particularly large with the wild-type form of alcohol dehydrogenase from horse liver. We analyze alcohol dehydrogenase catalysis through a re-examination of the reaction coordinates. The ground states of the binary and ternary complexes are shown to be related to the corresponding transition states through topology and free energy acting along the reaction path.