Isomerization of an enzyme-coenzyme complex in yeast alcohol dehydrogenase-catalysed reactions (original) (raw)
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Binding of coenzymes to yeast alcohol dehydrogenase
Journal of the Serbian Chemical Society, 2010
In this work, the binding of coenzymes to yeast alcohol dehydrogenase (EC 1.1.1.1) were investigated. The main criterions were the change in the standard free energies for individual reaction steps, the internal equilibrium constants and the overall changes in the reaction free energies. The calculations were performed for the wild type enzyme at pH 6-9 and for 15 different mutant type enzymes, with single or double point mutations, at pH 7.3. The abundance of theoretical and experimental data enabled the binding of coenzymes to enzyme to be assessed in depth.
Mechanism of the Alcohol Dehydrogenases from Yeast and Horse Liver
European Journal of Biochemistry, 1971
Studies of the alcohol-acetaldehyde interchange, in the presence of analogues of NAD+ and brought about by yeast and horse liver alcohol dehydrogenases, have not provided any evidence in favour of the direct participation of the enzyme in the hydrogen transfer step. A new preparation of 1,4,5,6-tetrahydro-nicotinamide adenine dinucleotide is described. This analogue has been found to be a good competitive inhibitor for both enzymes, thus demonstrating the importance of fixation of the enzyme by a hydrogen bond to the group present in C-3 of the nicotinamide nucleus.
International Journal of Science and Research Archive, 2024
Alcohol dehydrogenases (ADHs) are zinc-containing enzymes that catalyze the oxidation of alcohols to aldehydes or ketones. The enzymes also play a critical role in the metabolism of a number of drugs and metabolites containing alcohol functional groups. Kinetic parameters of yeast alcohol dehydrogenase (ADH) was estimated using spectrophotometer, optimum temperature and pH for ethanol, as well as ADH specificity to other substrates was determined. Results from the assay showed value for the initial velocity of reaction of the enzyme estimated as 0.297. Change in concentration of NADH which correspond to ADH activity for ethanol was calculated as 48nanokatals (4.8 X 10-5 mol-1 l), the results also showed average values of the Km and Vmax of ADH for ethanol as estimated from Michaelis-Menten and Line weaver-Burk double reciprocal plots, to be 21.5mM and 0.426 respectively. Optimum temperature and pH of ADH for ethanol were estimated to be 25 °C and 8 respectively. Although, reaction specificity of ADH to other substrates showed gradation between propan-2-ol and ethanol with ethanol showing highest activity and methanol least. 2-propene-1-ol also presented high enzyme activity relative to ethanol. In most of enzyme assays, enzyme activity is determined by measuring the rate of conversion of substrate or rate of production of products within a given period of time. In this experiment, the rate of oxidation of NADH was monitored since NADH has a known maximum light absorbance at 340nm.
Archives of biochemistry and biophysics, 2016
Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 Å where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 Å to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxyl...
Aldehyde dismutase activity of yeast alcohol dehydrogenase
1999
Yeast alcohol dehydrogenase (EC 1.1.1.1) is able to catalyze the oxidation of acetaldehyde by NAD + with a concomitant formation of ethanol, at pH 8.8 and pH 7.1; the stoichiometry of aldehyde oxidation vs. ethanol formation is 2:1. This enzymatic reaction obeys the Michaelis-Menten kinetics and was characterized by a high K M for acetaldehyde (68 mM) and a low k cat (2.3 s −1), at pH 8.8, 22 • C. There is no visible burst of NADH during the reaction, from pH 7.1-10.1. Therefore, we have concluded that the enzyme catalyzes an apparent dismutation of two molecules of acetaldehyde into a molecule of acetic acid and a molecule of ethanol.
Journal of Molecular Biology, 1999
Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coef®cients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD or the NAD-3-pentanone adduct. The k on velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD complex, with a ÁH ion value of 74(AE4) kJ/mol (18(AE1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pK a values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pK a values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.
Structure and function of yeast alcohol dehydrogenase
Journal of the Serbian Chemical Society, 2000
1. Introduction 2. Isoenzymes of YADH 3. Substrate specificity 4. Kinetic mechanism 5. Primary structure 6. The active site 7. Mutations in the yeast enzyme 8. Chemical mechanism 9. Binding of coenzymes 10. Hydride transfer This article has been corrected. Link to the correction 10.2298/JSC0008609E
The Protein Journal, 2008
A study has been made of the effect of sodium dodecylsufate (SDS) addition on the oxidation of ethanol catalyzed by yeast alcohol dehydrogenase. Experiments were performed at pH = 8.1 and SDS concentrations employed were below and above the surfactant critical micelle concentration (CMC). The double reciprocal plots obtained in the absence and in the presence of the surfactant were compatible with a sequential bi-bi ordered mechanism. In the presence of the surfactant the initial reaction rates were consistently lower than in pure buffer at all the surfactant concentrations considered (0.5-50 mM). This effect is mainly due to an increase in the dissociation constant of b-NAD + which reaches its maximum value (7,100 ± 1,700 lM) at the CMC. Above the CMC the effect of the surfactant is mainly due to an increase in the Michaels constants of the alcohol, with values of 41 ± 1 mM for 15 mM SDS and 50 ± 1 mM for 50 mM SDS. The catalytic rate constant was found to be practically independent of the presence of the surfactant in the range of concentrations considered (up to 50 mM).
European Journal of Biochemistry, 1995
The lack of crystal structure for tetrameric yeast alcohol dehydrogenases (ADHs) has precluded, until now, the identification of the residues involved in subunit contacts. In order to address this question, we have characterized the thermal stability and dissociation propensity of native ADH I and ADH I1 isozymes as well as of several chimeric (ADH I-ADH 11) enzymes. Three groups of substitutions affecting the thermostability have been identified among the 24 substitutions observed between isozymes I and 11. The first group contains a Cys277+Ser substitution, located at the interface between subunits in a threedimensional model of ADH I, based on the crystallographic structure of the dimeric horse liver ADH. In the second group, the Asp236-Asn substitution is located in the same interaction zone on the model. The stabilizing effect of this substitution can result from the removal of a charge repulsion between subunits. It is shown that the effect of these two groups of substitutions correlates with changes in dissociation propensities. The third group contains the Metl68+Arg substitution that increases the thermal stability, probably by the formation of an additional salt bridge between subunits through the putative interface. These data suggest that at least part of the subunit contacts observed in horse liver ADH are located at homologous positions in yeast ADHs.