Inhibition Studies on Liver Alcohol Dehydrogenase

Evans, N.J.; Rabin, B. R.

doi:10.1111/j.1432-1033.1968.tb00247.x

Cited by 46 publications

(30 citation statements)

References 18 publications

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“…from horse liver forms highly-fluorescent complexes with NADH and imidazole [ 1 ] . Enhancement of fluorescence of enzyme-bound NADH was used to estimate dissociation constants of binary and ternary complexes, at pH values of 6,7,8 and 9. lmidazole also stimulates inactivation of the enzyme by iodoacetate [2,31 ; dissociation constants of the binary enzyme-imidazole complex were calculated at pH 7.2 and 9.0, and 7.4 and 10.0, respectively. These two methods gave similar values in the pH region 7 ~ 8, but the discrepancy at pH 9 was considerable.…”

Section: Introductionmentioning

confidence: 99%

Evidence for two different complexes of liver alcohol dehydrogenase with imidazole

Reynolds¹,

McKinley-McKee²

1972

FEBS Letters

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Section: Introductionmentioning

confidence: 99%

Evidence for two different complexes of liver alcohol dehydrogenase with imidazole

Reynolds¹,

McKinley-McKee²

1972

FEBS Letters

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“…Evans and Rabin [2] showed that AMP and ADP protected liver alcohol dehydrogenase from iodoacetate. As shown in Figs.l and 2, protection by AMP, ADP, ATP, GMP and ADP-ribose is competitive with iodoacetate.…”

Section: Adenine Nucleotidesmentioning

confidence: 99%

Complexes of Liver Alcohol Dehydrogenase

Reynolds¹,

Morris²,

McKinley-McKee³

1970

European Journal of Biochemistry

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The Michaelis-Menten-type kinetics of inactivation of horse-liver alcohol dehydrogenase with iodoacetate have been used t o 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 a t p H 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. I n 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 zinc in the enzyme, and anions might bind to either, or neither.It has already been shown [1] that iodoacetate binds reversibly to horse-liver alcohol dehydrogenase, as well as inactivating it by alkylating two essential sulphydryl groups, i. e. one per subunit [2-41: a t ionic strength 0.1, various anions were found to protect the enzyme competitively with iodoacetate. I n this study, the work is extended to include nucleotides and zinc-binding agents known to bind to alcohol dehydrogenase. Multiple inhibition kinetics are used to examine the mutual effect of different pairs of ligands. Since these inactivation studies cannot be carried out satisfactorily a t pH 10 (in the absence of imidazole), substrate-kinetics studies are also used in some cases, to complement the inactivation studies. MATERIALS AND METHODSHorse-liver alcohol dehydrogenase was purchased as a crystalline suspension from Boehringer Mann- Nucleotides were purchased from Sigma Chemical Co. (St. Louis, Mo.) or P-L Biochemicals (Milwaukee, Wis.). Imidazole was recrystallised as described previously [6]. 4-Biphenyl-carboxylic acid was purchased from Fluka AG (Buchs, Switzerland) and recrystallised from methanol and then methanol-water : melting point 223-225". Other chemicals were of reagent grade.Inactivations were always carried out a t 23.5", a t constant ionic strength, maintained by adding phosphate where necessary. At p H 10, glycine buffer was used, concentration approximately 20 mM in glycinate anion. With imidazole present, hydrolysis of iodoacetate (found previously, a t high pH [I]) was negligible, as inactivation experiments were usually completed comparati...

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“…These neutral ligands may be producing conformational changes which affect the active site of the enzyme. The acceleration of the inactivation of the enzyme by iodoacetate in the presence of excess imidazole has been considered due to a conformational change induced by imidazole [28,4]. Such conformational changes may protect the active-centre lysine from pyridoxal-P.…”

Section: The Effect Of Coenzyme and Inhibitors On The Enhancement Of mentioning

confidence: 99%

The Lysines in Liver Alcohol Dehydrogenase

McKinley-McKee¹,

Morris²

1972

European Journal of Biochemistry

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Liver alcohol dehydrogenase is reversibly inactivated by pyridoxal 5'-phosphate. When the enzyme is saturated with pyridoxal-P the residual activity is about 2001,. The enzyme can be completely inactivated by successive covalent labelling with pyridoxal-P, using sodium borohydride reduction. A maximum of about 11 pyridoxal-P groups per subunit were covalently incorporated into the enzyme.The binding to the enzyme of several known inhibitors was investigated by studying their effect on the reversible inactivation of the enzyme by pyridoxal-P a t pH 8.0. The effect of these ligands on the activation of the enzyme by methyl picolinimidate was also studied. AMP protected competitively against inactivation by pyridoxal-P and also protected strongly against activation by methyl picolinimidate. After picolinimidylation of the enzyme the binding of AMP a t pH 10 was 8-fold weaker.AMP and NADH both protected one lysine residue per subunit from reaction with pyridoxal-P . It is concluded that there is one lysine residue a t the active centre of the enzyme and that this lysine plays an important part in binding the coenzyme by attracting the phosphate group of the AMP moiety of the coenzyme. Coenzyme binding to the picolinimidylated enzyme is weakened by steric interference between the picolinomidylated active-centre lysine and the AMP moiety of the coenzyme. Since effects of neutral zinc-binding ligands cannot be explained by steric or electrostatic effects, it is suggested that they produce conformational changes in the enzyme, perhaps by binding a t a site other than the active-centre zinc atom.

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Inhibition Studies on Liver Alcohol Dehydrogenase

Cited by 46 publications

References 18 publications

Evidence for two different complexes of liver alcohol dehydrogenase with imidazole

Evidence for two different complexes of liver alcohol dehydrogenase with imidazole

Complexes of Liver Alcohol Dehydrogenase

The Lysines in Liver Alcohol Dehydrogenase

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