Inactivation Of Yeast Alcohol Dehydrogenase Research Articles

Distinct symmetry and limited peptide refolding activity of the thermosomes from the acidothermophilic archaea Acidianus tengchongensis S5 T

Recombinant thermosomes from the Acidianus tengchongensis strain S5 T were purified to homogeneity and assembled in vitro into homo-oligomers (rATcpnα or rATcpnβ) and hetero-oligomers (rATcpnαβ). The symmetries of these complexes were determined by electron microscopy and image analysis. The rATcpnα homo-oligomer was shown to possess 8-fold symmetry while both rATcpnβ and rATcpnαβ oligomers adopted 9-fold symmetry. rATcpnαβ oligomers were shown to contain the α and β subunits in a 1:2 ratio. All of the complexes prevented the irreversible inactivation of yeast alcohol dehydrogenase at 55 °C and completely prevented the formation of aggregates during thermal inactivation of citrate synthase at 45 °C. All rATcpn complexes showed trace ATP hydrolysis activity. Furthermore, rATcpnβ sequestered fully chemically denatured substrates (GFP and thermophilic malic dehydrogenase) in vitro without refolding them in an ATP-dependent manner. This property is similar to previously reported properties of chaperonins from Sulfolobus tokodaii and Sulfolobus acidocaldarius. These features are consistent with the slow growth rates of these species of archaea in their native environment.

Stabilization of free and immobilized enzymes using hyperthermophilic chaperonin

Chaperonins suppress the denaturation of proteins and promote protein folding in vivo. Because hyperthermophilic chaperonins are expected to be used as a stabilizer for proteins, the effects of a group II chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1 (T. KS-1 cpn), on the stabilization of mesophilic and thermophilic free enzymes and an enzyme co-immobilized with T. KS-1 cpn were studied. T. KS-1 cpn prevented the thermal inactivation of yeast alcohol dehydrogenase (ADH), jack bean urease, and Thermus flavus malate dehydrogenase (MDH) at high temperatures. T. KS-1 cpn also improved the long-term stability of ADH at lower temperatures. Moreover, the residual ADH activity of ADH co-entrapped with T. KS-1 cpn was improved and maintained at a higher level than that of the entrapped ADH without chaperonin. T. KS-1 cpn is useful for the stabilization of free and immobilized enzymes and applicable to various fields of biotechnology.

Unusual Solute Protection Against Pressure Inactivation of Yeast Alcohol Dehydrogenase

A 3:1 combination of sucrose and glycine provides significantly greater protection against pressure-induced inactivation of yeast alcohol dehydrogenase than either solute alone. Trehalose, alone, gives much greater protection than sucrose alone, but not so in combination with glycine. These are striking new findings that cannot be accounted for by current theories of protein stabilization.

Effects of Trehalose on Pressure-Induced Inactivation of Yeast Alcohol Dehydrogenase

Isozymes of yeast alcohol dehydrogenase are slowly denatured at moderate hydrostatic pressures (<3 kbar). The time courses for inactivation are biphasic and both phases of both isozymes are protected by trehalose. ADH-I is slightly more barostable than ADH-II which is opposite to their thermostabilities. Trehalose at 1M extends their half-lives about 6-fold at 2 kbar, pH 7.5 and 25 degrees C. In contrast, 1M sucrose provides only 4.4-fold protection under identical conditions, a finding consistent with the superior protein stabilization of trehalose to other denaturants.

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 the course of inactivation of yeast alcohol dehydrogenase by 4-(2-pyridylazo)-resorcinol.

The kinetic theory of the substrate reaction during modification of enzyme activity previously described by Wang and Tsou has been applied to a study on the kinetics of the course of inactivation of alcohol dehydrogenase by 4-(2-pyridylazo)-resorcinol (PAR). The results showed that the inhibition of this enzyme by PAR was irreversible. A plot of 1n([P] infinity -[P]) versus give a straight line, suggesting that the inactivation kinetic course is monophasic. The kinetic analysis of the substrate reaction with different concentrations of the substrate and the inactivator has shown that the inactivation of yeast alcohol dehydrogenase by PAR involves complex formation, and that the substrate ethanol competes with PAR at the active site of the enzyme. The dissociation constants between the substrates and the enzyme as well as the microscopic rate constants for the inactivation of the enzyme have been determined.

Inactivation of Yeast Alcohol Dehydrogenase by Nitrilopropionamides

A series of halonitrilopropionamides have been examined as potential inhibitors of yeast alcohol dehydrogenase. Analogues with a good leaving group on the alpha-carbon, and a geminal electronegative atom, were found to be initial competitive inhibitors against NAD with inhibition constants as low as 0.6 microM. Incubation of the enzyme with these inhibitors leads to a slow, irreversible inactivation, with an inactivation constant for 2,2-dibromo-3-nitropropionamide of about 100 microM. No protection against inactivation was observed with the substrate ethanol, while the presence of saturating levels of NAD slowed the rate, but not the final extent, of enzyme inactivation. The resulting enzyme-inactivator complex is stable to a range of conditions that are known to denature the enzyme, indicating that a covalent modification of yeast alcohol dehydrogenase has led to the inactivation. A bimodal inactivation model is proposed to account for the observed interactions of these halonitrilopropionamides with yeast alcohol dehydrogenase.

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.

Inactivation of yeast alcohol dehydrogenase by alkylperoxyl radicals: Characteristics and influence of nicotinamide-adenine dinucleotides

The study of the interaction of alkylperoxyl radicals generated by the aerobic thermolysis of 2,2'-azobis(2-amidinopropane) (AAP) with yeast alcohol dehydrogenase (YADH) revealed a high reactivity of the enzyme, with an average of about 20 radicals per added YADH tetramer being needed to elicit its total inactivation. NAD + enhanced YADH inactivation at NAD +/YADH molar ratios from 0.25 to 1, decreasing the rate of the process when added in excess to the enzyme concentration. At NADH/YADH molar ratios greater than 1, NADH exhibited a protective effect characterized by a poorly defined induction time and lower inactivation rates, which progressively increased during the reaction period. These changes occurred concomitantly with the oxidation of NADH into NAD +, which might counteract the protective effect of NADH. Under similar conditions, NADP + did not modify AAP-induced YADH inactivation, while NADPH exhibited a modest protection at NADPH/YADH molar ratios greater than 1. It is concluded that YADH inactivation by alkylperoxyl radicals is strongly dependent on the redox state of the NADH-NAD + couple, as the rates of the process at different time intervals inversely correlate with the respective NADH/NAD + ratios.

Sulfhydryl enzyme inactivation by nicotinoylacrylates

Nicotinoylacrylic acid and the corresponding methyl, ethyl, propyl and benzyl esters were studied with respect to selective inactivation of dehydrogenases through covalent modification of essential sulfhydryl groups. An effective inactivation of yeast alcohol dehydrogenase, yeast glutathione reductase and yeast 6-phosphogluconate dehydrogenase was observed with methyl and ethyl nicotinoylacrylates, with nicotinoylacrylic acid and the larger propyl and benzyl esters being considerably less effective. Fluorescein mercuric acetate titration studies of inactivated yeast alcohol dehydrogenase and studies of the oxidized (disulfide) form of yeast glutathione reductase were consistent with inactivation processes involving sulfhydryl modification. Protection against nicotinoylacrylate inactivation was afforded by the binding of ligands to either the coenzyme or substrate binding sites. Inactivation of yeast alcohol dehydrogenase by ethyl nicotinoylacrylate exhibited an unexpected greater selectivity, resulting in the covalent modification of only one of the two reactive sulfhydryl groups at the catalytic site.

Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide

Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett. 40, 29-32). Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label. The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site. The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants. In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified. Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.

Inactivation of yeast alcohol dehydrogenase by 2-mercaptoethanol: Loss of zinc from the active centre

1. 1. Yeast alcohol dehydrogenase was inactivated by 2-mercaptoethanol (50 mM or greater). 2. 2. The commercial enzyme preparation used had 10.4g-atom zinc/molecule. Dialysis against ethylene-diaminetetraacetic acid solution lowered this value to 4.4 g-atom/molecule. 3. 3. Exposure to 500 mM 2-mercaptoethanol and subsequent dialysis lowered the zinc content to 1.4 g-atom/molecule, and caused a loss of enzyme activity (95%).

Studies of yeast alcohol dehydrogenase with 3-aminopyridine monucleotide.

3-Aminopyridine mononucleotide, a nicotinamide mononucleotide analog, was prepared by enzymatic cleavage of 3-aminopyridine adenine dinucleotide by a snake venom phosphodiesterase and isolated by means of ion exchange chromatography. The spectrophotometric and fluorometric properties of this analog were studied. Several anions were shown to quench the fluorescence intensity of this analog. pH was shown to have a pronounced effect on the fluorescence intensity. 3-Aminopyridine mononucleotide was shown to be a coenzyme-competitive inhibitor of yeast alcohol dehydrogenase. The 3-aminopyridine mononucleotide was diazotized with the use of nitrous acid. A time dependent irreversible inactivation of yeast alcohol dehydrogenase resulted from incubation with the diazotized 3-aminopyridine mononucleotide at pH 7.0. Incubation of the enzyme with NAD prior to the addition of the diazotized 3-aminopyridine mononucleotid protected the enzyme against inactivation.

The role of an essential histidine residue of yeast alcohol dehydrogenase.

1. Inactivation of yeast alcohol dehydrogenase for diethyl pyrocarbonate indicates that one histidine residue per enzyme subunit is necessary for enzymic activity. The inactivated enzyme regains its activity over a period of days. 2. Enzyme modified by diethyl pyrocarbonate can form the binary enzyme - NADH complex with the same maximum NADH-binding capacity as that of native enzyme. Modified enzyme cannot form normal ternary complexes of the type enzyme - NADH - acetamide and enzyme - NAD+ - pyrazole, which are characteristic of native enzyme. 3. The rate constant for the reaction of enzyme with diethyl pyrocarbonate has been determined over the pH range 5.5--9. The histidine residue involved has approximately the same pKa as free histidine, but is 10-fold more reactive than free histidine.

A novel diazonium-sulfhydryl reaction in the inactivation of yeast alcohol dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide.

Diazotized 3-aminopyridine adenine dinucleotide has been found to modify four sulfhydryl groups per molecule of enzyme during the complete inactivation of yeast alcohol dehydrogenase. The reaction of sulfhydryl groups was indicated by titration studies with 5,5-dithiobis(2-nitrobenzoic acid) as well as isolation and quantitation of the cysteinyl derivative released by acid hydrolysis of the modified enzyme. The cysteinyl derivative was identified as S-(3-pyridyl)cysteine. Authentic S-(3-pyridyl)cystein was synthesized and structurally characterized for these studies. Diazonium-sulfhydryl reactions were demonstrated for a number of diazonium derivatives with cysteine, homocysteine, glutathione, and mercaptoethanol at 0-4 degrees and neutral pH. Second order rate constants were determined in reactions of these sulfhydryl compounds with diazotized 1-methyl-3-aminopyridinium chloride, diazotized 3-aminopyridine adenine dinucleotide, and diazotized 3-aminopyridine adenine dinucleotide phosphate.

Measurements of the concentration of active sites in preparations of yeast alcohol dehydrogenase.

Studies on the inactivation of yeast alcohol dehydrogenase by iodoacetate indicate that under certain conditions reaction of two equivalents of reagent produces a 90–95% loss of activity. Such observations suggest two essential thiol groups per molecule. Measurements of NADH binding using three independent methods indicate a similar number of coenzyme binding sites.

Primary and secondary radicals in the radiation-induced inactivation of yeast alcoholdehydrogenase.

SummaryRadiation inactivation of YADH in dilute aqueous solution that does not contain oxygen is due to H atoms and OH radicals, whose inactivating efficiencies are in the ratio 9 : 1. The same species are responsible for the transient absorption arising in pulse-irradiated YADH in the region of 300–320 nm, and in this case their relative efficiencies are 7·5 to 1. This resulted from steady-state and pulse radiolysis experiments using radical scavengers such as t-butanol, formate and N2O. It appears that oxygen, when present, may also contribute to the enzyme inactivation through its radiation-induced radicals. Secondary radicals originated by reaction of OH radicals with CNS−, Br− and I−, all inactivate the enzyme at a faster rate than their primary parent radical. Their relative inactivating efficiencies are of the same order as their rate constants for reaction with YADH.

γ-Radiation Inactivation of Yeast Alcohol Dehydrogenase in the Presence of Oxidized Nicotinamide-Adenine Dinucleotide

γ-Radiation Inactivation of Yeast Alcohol Dehydrogenase in the Presence of Oxidized Nicotinamide-Adenine Dinucleotide

Ultrasonic Inactivation of Yeast Alcohol Dehydrogenase: Protection by Oxidized Nicotinamide-Adenine Dinucleotide

Conference Article| May 01 1973 Ultrasonic Inactivation of Yeast Alcohol Dehydrogenase: Protection by Oxidized Nicotinamide-Adenine Dinucleotide ALAN WISEMAN; ALAN WISEMAN 1Department of Biochemistry, University of Surrey, Guildford, Surrey, U.K. Search for other works by this author on: This Site PubMed Google Scholar PHILLIP H. LESLIE; PHILLIP H. LESLIE 1Department of Biochemistry, University of Surrey, Guildford, Surrey, U.K. Search for other works by this author on: This Site PubMed Google Scholar GEOFFREY C. GRANVILLE GEOFFREY C. GRANVILLE 1Department of Biochemistry, University of Surrey, Guildford, Surrey, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1973) 1 (3): 606–608. https://doi.org/10.1042/bst0010606 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation ALAN WISEMAN, PHILLIP H. LESLIE, GEOFFREY C. GRANVILLE; Ultrasonic Inactivation of Yeast Alcohol Dehydrogenase: Protection by Oxidized Nicotinamide-Adenine Dinucleotide. Biochem Soc Trans 1 May 1973; 1 (3): 606–608. doi: https://doi.org/10.1042/bst0010606 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1973 Biochemical Society1973 Article PDF first page preview Close Modal You do not currently have access to this content.

Inactivation of yeast alcohol dehydrogenase by dehydroascorbic acid and D-arabinosone.

Treatment of yeast alcohol dehydrogenase [EC 1.1.1.1] (YADH) with dehydroascorbic acid (DHA) or D-arabinosone resulted in inactivation of the enzyme. Effects of other dicarbonyl compounds, such as D-glucosone and triosone, on YADH were also examined, and they were found to be less effective than D-arabinosone or ineffective.The inactivation by DHA or D-arabinosone was accompanied by decrease of free sulfhydryl (SH) groups of the protein, and it was completely prevented by addition of SH compound. Treatment of the inactivated enzyme with β-mercaptoethanol led to partial reactivation with concomitant restoration of SH groups.The inactivation of YADH was accelerated by NAD+ as well as NADH specifically.These results strongly supported a possibility that DHA and D-arabinosone afforded the inhibitory effect on YADH through oxidation or modification of SH groups of the protein, which was favored in the presence of the coenzyme.