Fluorescence quenching of Trp-314 of liver alcohol dehydrogenase by oxygen

The quenching of the fluorescence of liver alcohol dehydrogenase (LADH) by molecular oxygen has been studied by both fluorescence lifetime and intensity measurements. This was done in the presence of 1 M acrylamide which selectively quenches the fluorescence of the surface tryptophan residue, Trp-15, thus allowing us to focus on the quenching of the deeply buried tryptophan, Trp-314, by molecular oxygen. Such studies yielded a Stern-Volmer plot of F0/F with a greater slope than the corresponding tau o/tau plot. This indicates that both dynamic and static quenching of Trp-314 occurs. The temperature dependence of the dynamic quenching of LADH by oxygen was also studied at three temperatures, from which we determined the activation enthalpy for the quenching of Trp-314 to be about 10 kcal/mol. The oxygen quenching of a ternary complex of LADH, NAD+ and trifluoroethanol was also studied. The rate constant for dynamic quenching of Trp-314 by oxygen was found to be approximately the same in the ternary complex as that in the unliganded enzyme.     

Singular oxygen effects on the room-temperature phosphorescence of alcohol dehydrogenase from horse liver.

The room-temperature phosphorescence of alcohol dehydrogenase from horse liver in the presence of oxygen has the characteristics of a light-induced emission as it appears only after a certain amount of excitation has been absorbed. The initial lag period, the steady-state intensity, and the duration of the induced state are dominated by the oxygen content of the solution, the rate of light absorption, and the radiation dose. The phenomenon is not unique to this enzyme and the data are consistent with photochemical depletion of oxygen.     

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.     

A new subunit of horse liver alcohol dehydrogenase and subunit composition of the polymorphic form.

The most cathodal (on starch-gel electrophoresis), steroid-active band of horse liver alcohol dehydrogenase, whose catalytic properties were shown to be dependent on the livers used as a starting material [Pietruszko (1974) Biochem. Biophys. Res. Commun. 60, 687-694], has been prepared from A-type and S-type horse livers by identical methods. Results presented here show that different isoenzymes are present in these preparations.     

Interaction of ethanolamine with alcohol dehydrogenase from the horse liver

The pattern of kinetic behaviour of ethanolamine (EA), an ethanol structural analog, in the alcohol dehydrogenase reaction has been studied. EA has been shown to manifest a mixed type inhibition versus ethanol and a noncompetitive behaviour towards the second substrate, NAD. A graphical analysis of the experimental results as well as the construction of secondary graphs provide evidence in favour of a mechanism, according to which the interaction between EA and the enzyme results in a dead-end complex formation (ESI). A direct conversion into reaction products can be achieved only after EA separation from the complex. The Ki value for the E-EA complex is 1.3 mM; that for EA release from the E-EA is 1.8 mM. An analysis of competitive interactions with NAD showed these constants to be equal in values (2 mM). Taking account of real concentrations of tissue EA and of experimental values of Ki, a conclusion is drawn on possible participation of EA in the alcohol dehydrogenase reaction control.     

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.     

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.     

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.     

Excitation transfer in complexes of horse liver alcohol dehydrogenase

The protein fluorescence of LADH¹ was quenched upon coupling with NADH, NAD⁺, o-phenanthroline, or thyroxine and its related compounds, while AMP, ADP, ADPR, or NMN did not quench the fluorescence. Addition of isobutyramide or pyrazole to E₂R₂ or E₂O₂ did not alter the degree of quenching. The coupling of two molecules of NADH to one molecule of E₂I₂ caused an equal fluorescence enhancement for both molecules of NADH when excited in its 340-mμ absorption band. However, with excitation in the protein absorption range, it was found that the binding of the first NADH molecule to LADH caused a larger fluorescence change than the binding of the second one. This was ascertained by following the increase of the fluorescence caused by addition of excess E₂ to E₂I₂R₂, whereby the complexes E₂I₂R and E₂I₂ were formed. This seemed to indicate that excitation energy could be transferred from one subunit to the other in the same LADH molecule.     

Cadmium-109 as a probe of the metal binding sites in horse liver alcohol dehydrogenase.

The noncatalytic and catalytic zinc atoms of horse liver alcohol dehydrogenase, [(LADH)Zn2Zn2] or LADH, have been replaced differentially with 109Cd by equilibrium dialysis, resulting in two new enzymatically active species, [(LADH)109Cd2Zn2] and [(LADH)109Cd2109Cd2]. The UV difference spectra of the cadmium enzymes vs. native [(LADH)Zn2Zn2] reveal maxima at 240 nm with molar absorptivities, delta epsilon 240, of 1.6 X 10(4) M-1 cm-1 per noncatalytic 109Cd atom and 0.9 X 10(4) M-1 cm-1 per catalytic 109Cd atom, consistent with coordination of the metals by four and two thiolate ligands, respectively, strikingly similar to the 250-nm charge-transfer absorbance in metallothionein. Carboxymethylation of the Cys-46 ligand to the catalytic metal in LADH presumably lowers the overall stability constant of the coordination complex and results in loss of catalytic 109Cd or catalytic cobalt but not catalytic zinc from the enzyme.     

Reactivity of horse liver alcohol dehydrogenase with 3-methylcyclohexanols

The specificity of horse liver alcohol dehydrogenase for cyclohexanol and its 3-methyl derivatives was investigated by stopped-flow and initial velocity kinetic studies. The (1S,3S)-3-methylcyclohexanol was 7 times more reactive (V/Km) than cyclohexanol, whereas the (1R,3R)-3-methylcyclohexanol was at least 1000 times less reactive than its enantiomer. Computer simulation of the transient reaction of NAD+ and the cyclohexanols catalyzed by the enzyme suggests that the rate of transfer of hydrogen from the alcohol to NAD+ is increased with the 1S,3S isomer. Modeling of the three-dimensional structure of the ternary complex of the enzyme suggests that the 1S,3S isomer should only bind in a productive, reactive mode, whereas the 1R,3R isomer would bind predominantly in a nonproductive, inhibitory mode.