Complexes Of Liver Alcohol Dehydrogenase Research Articles

Evidence for two different complexes of liver alcohol dehydrogenase with imidazole

Evidence for two different complexes of liver alcohol dehydrogenase with imidazole

Thermal stability of horse liver alcohol dehydrogenase and its complexes

Horse liver alcohol dehydrogenase (LADH) was protected against thermal denaturation by addition of NADH or NAD + and further protected by NADH + isobutyramide or NAD + + pyrazole. The effect was increased with increasing concentration of the ligand(s), indicating that the protection of LADH is dependent on equilibrium of the reaction between the enzyme and the ligand(s). The dissociation constants and the denaturation-rate constants of LADH complexes were determined from the thermal denaturation curves.

Binding and Kinetic Studies of Liver Alcohol Dehydrogenase-Coenzyme-Pyrazole Complexes

Complexes of liver alcohol dehydrogenase with pyrazole and NAD+, NHD+, or AcPyAD+ were studied by spectral, kinetic, and equilibrium binding techniques. Difference spectra showed the same peak, 290 mµ, for ternary complex with NAD+ or NHD+ as coenzyme but with AcPyAD+ as coenzyme the peak appeared at 308 mµ. The dissociation constants of coenzyme from the ternary complex were much higher for the two analogues than for NAD+. Stopped flow studies showed that for NHD+ the higher dissociation constant was due to a decreased on velocity of the coenzyme but for AcPyAD+ it was due to a higher off velocity than NAD+. The dissociation constants of pyrazole from the ternary complexes were the same for complexes with NAD+ and NHD+ but 8-fold higher for complex containing AcPyAD+. This was due to a much slower pyrazole on velocity. The results of this study indicate that pyrazole interacts with the nicotinamide ring of NAD+ in the ternary complex. The nature of this interaction is discussed.

Complexes of liver alcohol dehydrogenase. Further studies on the rate of inactivation.

The Michaelis‐Menten‐type kinetics of inactivation of horse‐liver alcohol dehydrogenase with iodoacetate have been used to study the binding of a number of known ligands. Adenine nucleotides protect competitively with iodoacetate as do halides and aromatic acids. Uncharged chelating‐agents (orthophenanthroline, and 2,2′‐bipyridyl) protect non‐competitively. Uncharged monodentate ligands (e. g. imidazole) stimulate inactivation.The effect of imidazole on the binding of various ligands at pH 7.4 and at pH 10 was studied. Multiple inhibition analysis was used to examine the mutual interaction of pairs of substances which protected the enzyme from iodoacetate. ADP‐ribose and decanoate were mutually competitive, while AMP and orthophenanthroline were non‐competitive. Increasing the ionic strength was found to affect differently the mutual interactions of pairs of anions, as well as to weaken their binding to the enzyme.In some cases, kinetic studies of reduction of NAD+ were used to complement the inactivation studies.It is concluded that, in the coenzyme, the phosphate group nearer to adenosine is important for binding. Most, but not all, of the ligand‐ligand interactions can be explained by steric or electrostatic effects; it is tentatively suggested that conformational changes may also be involved. Precise interpretation, however, is complicated: there are two different types of zine in the enzyme, and anions might bind to either, or neither.

Crystallization of Horse Liver Alcohol Dehydrogenase Complexes from Alcohol Solutions.

Crystallization of Horse Liver Alcohol Dehydrogenase Complexes from Alcohol Solutions.

Studies on liver alcohol dehydrogenase complexes: IV. Spectrophotometric observations on the enzyme complexes

The interaction of liver alcohol dehydrogenase with the coenzyme, adenosine diphosphate ribose (a coenzyme-competitive inhibitor) and substrate-competitive inhibitors such as caprate and isobutyramide was studied by means of double-difference spectrophotometry. Hitherto unreported changes in ultraviolet-light absorption were observed when the enzyme interacted with the coenzyme in the presence and absence of substrate-competitive inhibitors and with the adenine-nucleotide inhibitor. The coupling of the coenzyme and the adenine-nucleotide inhibitor to the enzyme caused an absorption increase with a maximum at 281 mμ and a decrease in the 260-mμ region. These and some other absorption changes were used for the direct determination of the dissociation constants of complexes of the enzyme. Newly determined were K E, R and K E, O at pH 10, and redetermined were K E,ADPR at pH 6–10 and K E-caprate, O . 1 1 Abbreviations used: DPNH (R), reduced diphosphopyridine dinucleotide; DPN (O), oxidized diphosphopyridine dinucleotide; LADH (E), liver alcohol dehydrogenase; ADPR, adenosine diphosphate ribose; ADP, adenosine diphosphate; AMP, adenosine monophosphate; and (I), substratecompetitive inhibitors. Letters in parentheses are simplified abbreviations used in expressing dissociation constants and forms of the enzyme complexes.

Studies on liver alcohol dehydrogenase complexes: III. Multiple inhibition kinetics in the presence of two competitive inhibitors

A graphical method was devised to analyze the multiple inhibition of an enzyme by two competitive inhibitors. This method not only distinguishes whether two inhibitors interact with the same site or different sites of the enzyme, but also gives an interaction constant (α) between two inhibitors in the enzyme-inhibitor complex. This method was applied to the multiple inhibition of liver alcohol dehydrogenase by coenzyme-competitive inhibitor pairs among o-phenanthroline, adenosine diphosphate ribose, and adenosine-5′-mono- and -diphosphates. The present kinetic analysis quantitatively reconfirmed our previous conclusion that o-phenanthroline and adenosine diphosphate ribose interact independently with different sites of the enzyme.

Spectrophotofluorometric Determination of the Dissociation Constants of Amides from the Enzyme-Reduced Coenzyme Complex of Liver Alcohol Dehydrogenase.

Spectrophotofluorometric Determination of the Dissociation Constants of Amides from the Enzyme-Reduced Coenzyme Complex of Liver Alcohol Dehydrogenase.

Spectrophotofluorometric Determination of the Dissociation Constants of Relatively Nonfluorescent Liver Alcohol Dehydrogenase Complexes with Amides and Coenzyme.

Spectrophotofluorometric Determination of the Dissociation Constants of Relatively Nonfluorescent Liver Alcohol Dehydrogenase Complexes with Amides and Coenzyme.

On the ternary complex of liver alcohol dehydrogenase with reduced coenzyme and isobutyramide. Effect of p-chloromercuriphenyl sulfonate and stability of the complex

The reaction between p-chloromercuriphenyl sulfonate and SH groups of the ternary complex of liver alcohol dehydrogenase, isobutyramide, and reduced coenzyme was studied by a new spectrophotofluorometric method. This is based upon the fact that reduced coenzyme is released from the ternary complex in proportion to the amount of mercurial added. Because of the high sensistivity of the method, SH groups of the enzyme in submicronormal (μ N) concentrations can be readily determined. Since these determinations can be carried out in the same cuvette following coenzyme-binding site titrations, the ratio of SH groups to coenzyme-binding sites can be determined with great accuracy: the ratio was found to be 14.1 and was independent of the purity of the enzyme preparations. In a mixture of free-enzyme binary and ternary complexes, the mercurial reacts preferentially with the enzyme in forms other than the ternary complex, indicating that the latter is more resistant to the mercurial reaction than the others. A strongly increased stability of the ternary complex is also demonstrated in acid, heat, and alkali inactivation experiments. The complex is completely stable under conditions in which free enzyme or binary complexes are considerably destroyed.

Two ternary complexes of liver alcohol dehydrogenase with reduced diphosphopyridine nucleotide and the inhibitor imidazole

Two ternary complexes of liver alcohol dehydrogenase with reduced diphosphopyridine nucleotide and the inhibitor imidazole