Inhibition Of Yeast Alcohol Dehydrogenase Research Articles

The competitive inhibition of yeast alcohol dehydrogenase by 2,2,2-trifluoroethanol

An experiment for undergraduate biochemistry laboratory classes demonstrating the competitive inhibition of yeast alcohol dehydrogenase by 2,2,2-trifluoroethanol is described. The experiment uses a convenient, rapid spectrophotometric assay and can be accomplished in one laboratory period. Students can measure the Michaelis-Menten constant for ethanol and determine the inhibition constant for 2,2,2-trifluoroethanol.

Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by 4,4′-dithiodipyridine

The course of inactivation of yeast alcohol dehydrogenase (YADH) using 4,4′-dithiodipyridine (DSDP) has been studied in this paper. The results show that the reaction mechanism between DSDP and YADH is a competitive, complexing inhibition. The microscopic constants for the inactivation of the free enzyme and the enzyme-substrate complex were determined. The presence of the substrate NAD + offers strong protection for this enzyme against inactivation by DSDP. The above results suggest that two Cys residues are essential for activity and are situated at the active site. These essential Cys residues should be Cys-46 and Cys-174 which are ligands to the catalytic zinc ion. Another Cys residue, which can be modified by DSDP, is non-essential for activity of the enzyme.

Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by o-phthaldehyde.

The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously has been applied to a study on the kinetics of the course of inactivation of yeast alcohol dehydrogenase (YADH) by o-phthaldehyde (OPTA). The microscopic constants for the reaction of the inactivators with the free enzyme and with the enzyme-substrate complexes were determined. The inactivation is a monophasic pseudo-first-order reaction with OPTA. The apparent rate constant A is independent of the OPTA concentration, indicating that the inactivation is a noncomplexing inhibition. The marked protective effect of substrates on the inactivation of YADH by OPTA has been observed. This result suggests that the modification of the enzyme by OPTA may occur at the active site.

Inhibition of ethanol and acetate metabolism in mice by chlordimeform and related compounds

1. Since chlordimeform or N′-(4-chloro- o-toly l)- N, N-dimethylformamidine and certain of its mammalian metabolites prolong ethanol induced sleep in mice, their effect on ethanol and acetate metabolism was examined. 2. Intraperitoneal treatment of mice with chlordimeform (50 mg/kg) and demethylchlordimeform or N′-(4-chloro- o-tolyl)- N-methylformamidine (50 mg/kg), the two most potent sleep inducers, appreciably inhibited the metabolism of ethanol to 14CO 2. Another metabolite, 4-chloro- to-formotoluidide (100 mg/kg), a very weak sleep inducer, also markedly inhibited ethanol metabolism. 3. Chlordimeform, demethylchlordimeform, and 4-chloro- o-formotoluidide inhibited the conversion of ethanol to 14CO 2 and the conversion of acetate to 14CO 2 in vitro by mouse liver preparations. 4. The potency of 4-chloro- o-formotoluidide as an inhibitor of acetate metabolism in vitro compared favorably with that of SKF-525A, a known inhibitor of microsomal mixed function oxidases. 5. At a concentration of 1 × 10 −3 M, demethylchlordimeform yielded 27.4% inhibition of yeast alcohol dehydrogenase as compared to 46.3% for thiourea, a known inhibitor of this enzyme. The other compounds gave little, if any, inhibition of yeast alcohol or aldehyde dehydrogenase. 6. Although it is not possible to explain the mechanism for ethanol sleep enhancement in mice by chlordimeform and its metabolite demethylchlordimeform, inhibition of the alcohol dehydrogenase by demethylchlordimeform may be significant in this connection. 7. The decrease in ethanol metabolism apparently was due in large part to the activity of chlordimeform, demethylchlordimeform and especially 4-chloro- o-formotoluidide as inhibitors of microsomal mixed function oxidases.

Inhibition by ethanol, acetaldehyde and trifluoroethanol of reactions catalysed by yeast and horse liver alcohol dehydrogenases

1. Produced inhibition by ethanol of the acetaldehyde-NADH reaction, catalysed by the alcohol dehydrogenases from yeast and horse liver, was studied at 25 degrees C and pH 6-9. 2. The results with yeast alcohol dehydrogenase are generally consistent with the preferred-pathway mechanism proposed previously [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. The observed hyperbolic inhibition by ethanol of the maximum rate of acetaldehyde reduction confirms the existence of the alternative pathway involving an enzyme-ethanol complex. 3. The maximum rate of acetaldehyde reduction with horse liver alcohol dehydrogenase is also subject to hyperbolic inhibition by ethanol. 4. The measured inhibition constants for ethanol provide some of the information required in the determination of the dissociation constant for ethanol from the active ternary complex. 5. Product inhibition by acetaldehyde of the ethanol-NAD+ reaction with yeast alcohol dehydrogenase was examined briefly. The results are consistent with the proposed mechanism. However, the nature of the inhibition of the maximum rate cannot be determined within the accessible range of experimental conditions. 6. Inhibition of yeast alcohol dehydrogenase by trifluoroethanol was studied at 25 degrees C and pH 6-10. The inhibition was competitive with respect to ethanol in the ethanol-NAD+ reaction. Estimates were made of the dissociation constant for trifluoroethanol from the enzyme-NAD+-trifluoroethanol complex in the range pH6-10.

Inhibition of yeast alcohol dehydrogenase by dehydroretronecine

The interaction of dehydroretronecine, a hepatocarcinogenic metabolite of the pyrrolizidine alkaloid monocrotaline, with yeast alcohol dehydrogenase (ADH), cysteine and bovine serum albumin (BSA) was studied. Dehydroretronecine inhibited ADH with an inhibition constant ( K i ) of 3.38 × 10 −2 m at pH 7.5 and 25 °C. No inhibitory effect was observed when excess cysteine was added to the assay system. The interaction of dehydroretronecine with cysteine was demonstrated by spectrophotometric titration. Titration of dehydroretronecine alone at 240 nm showed an apparent pK value of 5.0. whereas titration of dehydroretronecine in the presence of excess cysteine resulted in an increase of pK from 5.0 to 8.5. Equilibrium dialysis data indicated appreciable binding of dehydroretronecine to BSA at pH 4.2 and 6°C.

Borate inhibition of yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase is inhibited competitively by borate with respect to NAD+. An unusual mechanism of competitive inhibition prevails: the competition for the substrate NAD+ by borate and enzyme. The following evidence supports this conclusion. (1) Much greater inhibition is observed with respect to NAD+ as compared with NADH as substrates. (2) Borate decreases the equilibrium constant of the overall reaction in the direction of ethanol oxidation, therefore, borate enters directly into the overall reaction rather than merely decreases the effectiveness of the catalyst. (3) The Ki values for unrelated enzyme reactions are identical for NAD+. (4) Stopped-flow experiments show burst kinetics only when NAD+ and borate are not premixed. (5) The Ki value is identical with the inverse of the borate-NAD+ complexation constant. (6) The pH dependence of the inhibitor demonstrates that only the B(OH)4-species is inhibiting. These results are consistent with the preferable binding of borate to NAD+ as compared with NADH. These two binding constants were found to be equal to 2000 +/- 60 and 130 +/- 8 M-1, respectively. In contrast to the liver enzyme, the yeast enzyme does not show pre-steady-state burst reactions in the reduction of NAD+. This would indicate that the interconversion of ternary complexes is at least partially rate limiting for the yeast enzyme.

Allyl alcohol-induced irreversible inhibition of yeast alcohol dehydrogenase

Allyl alcohol-induced irreversible inhibition of yeast alcohol dehydrogenase

Inhibition of alcohol and lactic dehydrogenases by patulin and penicillic acid in vitro

The inhibition of yeast alcohol dehydrogenase (ADH) and rabbit-muscle lactic dehydrogenase (LDH) by the mycotoxins patulin and penicillic acid has been studied in vitro. Both mycotoxins were non-competitive inhibitors of ADH, but were competitive with regard to LDH. The inhibition constants ( K 1 ) of patulin and penicillic acid, respectively, were found to be 50 × 10 −5 m and 11 × 10 −4 m for ADH and 6·2 × 10 −6 mand 7·2 × 10 −5 mfor LDH. Cysteine reversed the inhibitory effect of both mycotoxins on LDH but not on ADH. The interaction of the thiol groups at the active site of the dehydrogenases with both mycotoxins is discussed.

Pyrazole-induced inhibition of yeast alcohol dehydrogenase

Pyrazole-induced inhibition of yeast alcohol dehydrogenase

Applications of enzyme-catalysed reactions in trace analysis—VI: Determination of mercury and silver by their inhibition of yeast alcohol dehydrogenase

The inhibition of yeast alcohol dehydrogenase is applied to the determination of 2–200 ng of mercury(II) and 1–100 ng of silver. The enzyme, when used to catalyse the oxidation of ethanol, or the reverse reaction, is equally sensitive to inhibitors.

Inhibition of yeast alcohol dehydrogenase by alkylating agents.

1 The kinetic parameters for the irreversible inhibition of yeast alcohol dehydrogenase by iodoacetate, bromopyruvate and N-ethylmaleimide have been measured. 2 For iodoacetate and bromopyruvate, the rate of inhibition decreases with increasing pH. It is concluded that the reaction is promoted by a positive centre on the enzyme with a pKa value of 7.2. It is suggested this is a zinc-hydrate. 3 The reaction of N-ethylmaleimide with the enzyme is first order in each component. Co-enzymes protest the protein against inhibition and dissociation constants of the enzyme-coenzyme complex have been computed. The rate of inhibition increases with increasing pH, inflecting about a pKa value of 8.2.

The inhibition of yeast alcohol dehydrogenase by 2-bromo-2-phenylacetaldehyde

The inhibition of yeast alcohol dehydrogenase by 2-bromo-2-phenylacetaldehyde

Multiple inhibition of yeast alcohol dehydrogenase by heterocyclic nitrogen bases

Multiple inhibition studies were carried out with inhibitor pairs composed of various combinations of eleven nitrogen bases previously shown to be competitive inhibitors with respect to NAD + in the yeast alcohol dehydrogenase (alcohol:NAD + oxidoreductase E.C.I.I.I.I.)-catalyzed oxidation of ethanol. In studies of inhibitor pairs in which both inhibitors were adenine derivatives, mutual exclusion was demonstrated. A mutual exclusion was likewise demonstrated in all combinations of inhibitors studied involving phenanthrolines and benzoquinolines. Quinoline, although bound simultaneously to the yeast enzyme with adenine derivatives, shows mutual exclusion when studied with N 1-methylnicotinamide chloride. It was proposed that phenanthrolines and benzoquinolines like quinoline are bound at the “pyridinium ring” region of the NAD + binding site of yeast alcohol dehydrogenase. Charge transfer complexes were observed between N 1-methylnicotinamide chloride and the four adenine derivatives studied.

Nitrogen base inhibition of yeast alcohol dehydrogenase.

Summary The oxidation of ethanol as catalyzed by yeast alcohol dehydrogenase (alcohol: NAD + oxidoreductase, EC 1.1.1.1) is inhibited by a variety of nitrogen bases. Inhibition by 2,9-dimethyl-1,10-phenanthroline, 1,5-phenanthroline, 5,6-benzoquinoline, 7,8-benzoquinoline, quinoline and adenosine was in each case shown to be competitive with respect to NAD + in these reactions. With the exception of adenosine, these nitrogen bases are bound to the enzyme more effectively than is 1,10-phenanthroline. The data indicate that the binding of phenanthrolines, benzoquinolines and quinoline to the enzyme occurs at the pyridinium ring region of the NAD + binding site and is stabilized through hydrophobic interactions rather than by zinc chelation.

Multiple inhibition of yeast alcohol dehydrogenase

Multiple inhibition of the yeast alcohol dehydrogenase-catalyzed oxidation of ethanol was investigated; four different inhibitors were used that were known to function competitively with respect to NAD + in these reactions. The inhibitors, adenosine diphosphate ribose, adenosine diphosphate, N 1-methylnicotinamide chloride, and 1,10-phenanthroline, were studied in pairs to determine the extent to which interactions between inhibitors occur on the enzyme. The inhibitor pairs showing a maximum interaction or mutual exclusion were adenosine diphosphate riboseadenosine diphosphate, N 1-methylnicotinamide chloride-1,10-phenanthroline, adenosine diphosphate ribose-1,10-phenanthroline, adenosine diphosphate-1,10-phenanthroline, and N 1-methylnicotinamide chloride-adenosine diphosphate. A slight interaction was observed when the pair, adenosine diphosphate ribose- N 1-methylnicotinamide chloride was studied. Multiple inhibition studies with 1,10-phenanthroline were consistent with the binding of this inhibitor through interactions with the pyridinium site and the hydrophobic region of the enzyme.

Inhibition of yeast alcohol dehydrogenase by alkylammonium chlorides

Five alkylammonium chlorides were shown to be competitive inhibitors with respect to NAD+ in the yeast alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1)-catalyzed oxidation of ethanol. This inhibition increases with increasing chain length of the alkyl substituents of these compounds and is consistent with a hydrophobic interaction between these compounds and yeast alcohol dehydrogenase. In the reverse reaction, the reduction of acetaldehyde, the alkylammonium chlorides enhance enzyme activity at low concentrations and inhibit enzyme activity at high concentrations. These effects also increase with increasing chain length of the alkyl substituents of these compounds.

Potassium sorbate inhibition of yeast alcohol dehydrogenase

Potassium sorbate was found to inhibit yeast alcohol dehydrogenase (alcohol: NAD oxidoreductase, EC 1.1.1.1) maximally at the pH optimum of the enzyme; viz. pH 8.5. Potassium sorbate competed with NAD and ethanol for a site on the enzyme but in an irreversible manner. Sorbamide was found to be approx. 1000 times a better inhibitor of yeast alcohol dehydrogenase than was potassium sorbate. The evidence presented indicates that potassium sorbate inhibits yeast alcohol dehydrogenase irreversibly by either the formation of a covalent bond between the sulfur of the essential sulfhydryl group or the ZnOH − of the enzyme and the δ and/or β carbons of the sorbate ion.

The inhibition of yeast alcohol dehydrogenase by borate

The inhibition of yeast alcohol dehydrogenase by borate

Adventitious inhibition of yeast alcohol dehydrogenase

The inhibition of yeast alcohol dehydrogenase by N′-methylnicotinamide has been shown to be due to contaminating silver ions present in commercial preparations of this reagent. Contamination by metal ions, which was assessed by quantitative analysis, also accounts for the inhibition of yeast alcohol dehydrogenase by semicarbazide. Purification of the reagents by removal of the metals abolished the inhibition. The existence of enzyme concentration-dependent inhibition serves as a kinetic indicator of such an adventitious inhibition.