A study of the pH- and temperature-dependence of the reactions of yeast alcohol dehydrogenase with ethanol, acetaldehyde and butyraldehyde as substrates

The kinetics of ethanol oxidation by NAD+, and acetaldehyde and butyraldehyde reduction by NADH, catalysed by yeast alcohol dehydrogenase, were studied in the pH range 4.9--9.9 at 25 degrees C and in the temperature range 14.8--43.5 degrees C at pH 7.05. The kinetics of reduction of acetaldehyde by [4A-2H]NADH at pH 7.05 and pH 8.9 at 25 degrees C were also studied. The results of the kinetic experiments indicate that the mechanism of catalysis, previously proposed on the basis of studies at pH 7.05 and 25 degrees C (Dickinson & Monger, 1973), applies over the wide range of conditions now tested. Values of some of the initial-rate parameters obtained were used to deduce information about the pH- and temperature-dependence of the specific rates of combination of enzyme and coenzymes and of the dissociation of the enzyme--coenzyme compounds. Primary and secondary plots of initial-rate data are deposited as Supplementary Publication SUP 50043 (20 pages) with the British Library (Lending Division), Boston Spa, Wetherby, Yorks. LS23 7BQ, U.K., from whom copies may be obtained under the terms indicated in Biochem. J. (1975) 145, 5.

Download Full-text

A study of the oxidation of butan-1-ol and propan-2-ol by nicotinamide-adenine dinucleotide catalysed by yeast alcohol dehydrogenase.

Biochemical Journal ◽

10.1042/bj1470541 ◽

1975 ◽

Vol 147 (3) ◽

pp. 541-547 ◽

Cited By ~ 15

Author(s):

C J Dickenson ◽

F M Dickinson

Keyword(s):

Alcohol Dehydrogenase ◽

Ternary Complexes ◽

Kinetics Of Oxidation ◽

Yeast Alcohol Dehydrogenase ◽

Rate Limiting Step ◽

Limiting Step ◽

Ph Range ◽

Rate Limiting ◽

Kinetics Of ◽

Flow Experiments

1. The kinetics of oxidation of butan-1-ol and propan-2-ol by NAD+, catalysed by yeast alcohol dehydrogenase, were studied at 25 degrees C from pH 5.5 to 10, and at pH 7.05 from 14 degrees to 44 degrees C, 2. Under all conditions studied the results are consistent with a mechanism whereby some dissociation of coenzyme from the active enzyme-NAD+-alcohol ternary complexes occurs, and the mechanism is therefore not strictly compulsory order. 3. A primary 2H isotopic effect on the maximum rates of oxidation of [1-2H2]butan-1-ol and [2H7]propan-2-ol was found at 25 degrees C over the pH range 5.5-10. Further, in stopped-flow experiments at pH 7.05 and 25 degrees C, there was no transient formation of NADH in the oxidation of butan-1-ol and propan-2-ol. The principal rate-limiting step in the oxidation of dependence on pH of the maximum rates of oxidation of butan-1-ol and propan-2-ol is consisten with the possibility that histidine and cysteine residues may affect or control catalysis.

Download Full-text

Kinetic effect of some aliphatic amines on yeast alcohol dehydrogenase

Canadian Journal of Biochemistry ◽

10.1139/o76-062 ◽

1976 ◽

Vol 54 (5) ◽

pp. 432-437

Author(s):

M. J. Dove ◽

C. S. Tsai

Keyword(s):

Alcohol Dehydrogenase ◽

Ethanol Oxidation ◽

Initial Rate ◽

Relative Concentration ◽

Aliphatic Amines ◽

Kinetic Effect ◽

Yeast Alcohol Dehydrogenase ◽

Protonated Amines ◽

Ph Range

Initial rate studies of ethanol oxidation catalyzed by yeast alcohol dehydrogenase (EC 1.1.1.1) were carried out in the presence of varying concentrations of aliphatic amines over the pH range from 8.0 to 10.5. Aliphatic amines either activate or inhibit the enzyme depending on whether the pH is greater or less than 9.5 suggesting that the protonated amines activate and the nonprotonated amines inhibit the enzyme. Aliphatic amines activate yeast alcohol dehydrogenase by decreasing Kb while they inhibit the enzyme by increasing both Ka and Kia. When both protonated and nonprotonated amines are present in solution, either overall activation or inhibition will be observed depending on the relative concentration of the two amine species.

Download Full-text

A study of the ionic properties of the essential histidine residue of yeast alcohol dehydrogenase in complexes of the enzyme with its coenzymes and substrates

Biochemical Journal ◽

10.1042/bj1610073 ◽

1977 ◽

Vol 161 (1) ◽

pp. 73-82 ◽

Cited By ~ 18

Author(s):

C J Dickenson ◽

F M Dickinson

Keyword(s):

Alcohol Dehydrogenase ◽

Initial Rate ◽

Dissociation Constants ◽

Ternary Complexes ◽

Histidine Residue ◽

Mechanism Of Formation ◽

Binary And Ternary Complexes ◽

Yeast Alcohol Dehydrogenase ◽

Ph Range

1. Initial-rate studies of the reduction of acetaldehyde by NADH, catalysed by yeast alcohol dehydrogenase, were performed at pH 4.9 and 9.9, in various buffers, at 25 degrees C. The results are discussed in terms of the mechanism previously proposed for the pH range 5.9-8.9 [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. 2. Acetaldehyde forms a u.v.-absorbing complex with glycine. This was shown not to affect the results of kinetic experiments under the conditions used in this and earlier work. 3. The variation with pH of the dissociation constant for the enzyme-NADH complex, calculated from the initial-rate data, indicates that the enzyme possesses a group with pK7.1 in the free enzyme and pK8.7 in the complex. 4. The pH-dependences of the second-order rate constants for inactivation of the enzyme by diethyl pyrocarbonate were determined for the free enzymes (pK7.1), the enzyme-NAD+ complex (pK approx. 7.1) and the enzyme-NADH complex (pK approx. 8.4). The essential histidine residue may therefore be the group involved in formation and dissociation of the enzyme-NADH complex. 5. Estimates of the rate constant for reaction of acetaldehyde with the enzyme-NADH complex indicate that acetaldehyde may combine only when the essential histidine residue is protonated. The dissociation constants for butan-1-ol and propan-2-ol, calculated on the basis of earlier kinetic data, are, however, independent of pH. 6. The results obtained are discussed in relation to the role of the essential histidine residue in the mechanism of formation of binary and ternary complexes of the enzyme with its coenzymes and substrates.

Download Full-text

Untersuchungen zur Reaktion der Alkoholdehydrogenase mit Tritium-markierten Substraten

Zeitschrift für Naturforschung B ◽

10.1515/znb-1966-0609 ◽

1966 ◽

Vol 21 (6) ◽

pp. 540-546 ◽

Cited By ~ 8

Author(s):

Dieter Palm

Keyword(s):

Alcohol Dehydrogenase ◽

Temperature Dependence ◽

Isotope Effect ◽

Substrate Binding ◽

Isotope Effects ◽

Direct Interaction ◽

Enzyme Complex ◽

Temperature Range ◽

Yeast Alcohol Dehydrogenase ◽

Rate Controlling Step

Unexpectedly, the isotope effect of ethanol-1-Τ as a substrate of yeast alcohol dehydrogenase, increases with rising temperature from kH/kT = 3.2 at 5 —15°C to 3.8—4.7 at 20 —35 °C. This suggests a change of the rate controlling step as proposed by MÜLLER-HILL and WALLENFELS, who investigated the temperature dependence of the activation energies in this temperature range. A comparison of the affinities of propanol and butanol with the isotope effects of the corresponding tritium labelled compounds (propanol-1-Τ 6.7 at 25 °C, butanol-1-Τ 6.8 at 25 °C) supports the proposal, that during substrate binding, there must be a direct interaction between the enzyme complex and hydrogen which is removed in the reaction. These influences are less pronounced for the ethanol homologues which are bound less tightly to the enzyme. Therefore the H transfering step proper gives a greater contribution to the overall experimental isotope effect.

Download Full-text

Über die pH- und Temperatur-Abhängigkeit des thermischen Zerfalls von Poliovirus-Teilchen in Nucleinsäure und leere Proteinhüllen

Zeitschrift für Naturforschung B ◽

10.1515/znb-1966-0410 ◽

1966 ◽

Vol 21 (4) ◽

pp. 357-361 ◽

Cited By ~ 7

Author(s):

O. Drees ◽

K. D. Demme

Keyword(s):

Heat Treatment ◽

Initial Rate ◽

Concentrated Suspensions ◽

Virus Particles ◽

Ph Values ◽

Intact Virus ◽

Wide Range ◽

Ultracentrifugal Analysis ◽

Increasing Temperature ◽

Kinetics Of

The intact virus particles of highly purified, concentrated suspensions of poliovirus have been disintegrated into free nucleic acid and empty protein shells (78 S protein) by moderate heat treatment at various temperatures and pH values. The kinetics of this degradation has been followed by ultracentrifugal analysis.With increasing temperature between 35° and 50 °C and with increasing pH of the suspension medium between 7 and 8, the rate of degradation increased. For any particular conditions the initial rate was not maintained, and after a certain time there was little further degradation if conditions remained unaltered. Some particles with the sedimentation characteristics of poliovirus were resistant to disintegration under the same conditions which led to the breakdown of the bulk of the virus. The proportion of this “stable fraction” varied within a wide range from one preparation to another and decreased with increasing temperature and with increasing pʜ.

Download Full-text

Kinetics of the Course of Inactivation of Yeast Alcohol Dehydrogenase by 4–(2-Pyridylazo)-Resorcinol

Journal of Enzyme Inhibition ◽

10.3109/14756369609036531 ◽

1996 ◽

Vol 10 (4) ◽

pp. 239-250 ◽

Cited By ~ 1

Author(s):

Ying-Xia Zhang ◽

Hai-Meng Zhou

Keyword(s):

Alcohol Dehydrogenase ◽

Yeast Alcohol Dehydrogenase ◽

Kinetics Of

Download Full-text

A study of the kinetics and mechanism of rabbit muscle l-glycerol 3-phosphate dehydrogenase

Biochemical Journal ◽

10.1042/bj1430019 ◽

1974 ◽

Vol 143 (1) ◽

pp. 19-27 ◽

Cited By ~ 14

Author(s):

Philip Bentley ◽

F. Mark Dickinson

Keyword(s):

Basic Mechanism ◽

Kinetics And Mechanism ◽

Dihydroxyacetone Phosphate ◽

Rabbit Muscle ◽

Kinetics Of Oxidation ◽

Mechanism Of Catalysis ◽

Glycerol 3 Phosphate Dehydrogenase ◽

Ph Range ◽

Kinetics Of

1. The kinetics of oxidation of l-glycerol 3-phosphate by NAD+and of reduction of dihydroxyacetone phosphate by NADH catalysed by rabbit muscle glycerol 3-phosphate dehydrogenase were studied over the range pH6–9. 2. The enzyme was found to catalyse the oxidation of glyoxylate by NAD+at pH8.0 and the kinetics of this reaction were also studied. 3. The results are consistent with a compulsory mechanism of catalysis for glycerol 3-phosphate oxidation and dihydroxyacetone phosphate reduction in the intermediate regions of pH, but modifications to the basic mechanism are required to fully explain results at the extremes of the pH range, with these substrates and for glyoxylate oxidation at pH8.0.

Download Full-text

Ruthenium and iron complexes of dipicolinic acid: Synthesis, solution properties and kinetics of electron transfer reactions with ascorbate ions

Australian Journal of Chemistry ◽

10.1071/ch9832377 ◽

1983 ◽

Vol 36 (12) ◽

pp. 2377 ◽

Cited By ~ 22

Author(s):

NH Williams ◽

JK Yandell

Keyword(s):

Redox Potential ◽

Rate Constant ◽

Dipicolinic Acid ◽

Acid Synthesis ◽

Equilibrium Mixture ◽

A Value ◽

Inert Electrode ◽

Ph Range ◽

Kinetics Of ◽

Kinetics Of Reduction

Standard potentials of the redox couples [bis(pyridine-2,6-dicarboxylate)MIII]-/2- ([M(dipic)2]-/2-, where M = Fe, Ru, Co) have been determined at 25�C, and ionic strength 0.1M (NaClO4 or KNO3). Kinetics of reduction of the oxidized complexes by ascorbate have also been examined under the same conditions. The [Fe(dipic)2]-/2- potential was found to be 355 � 5 mV. Reduction of [Fe(Fe(dipic)2]- in the pH range 4-6 was shown to occur by reaction with ascorbate monoanion (HA-) with a rate constant of (2.2 � 0.2) × 103 1. mol-1 s-1, and ascorbate dianion(A2-) with a rate constant of (7 � 1) × 108 1. mol-1 s-1. K [Ru(dipic)2] has been synthesized. Spectroscopic and analytical evidence suggest that it is a simple six-coordinate species in the solid and in non-aqueous solvents, but that in water it exists as an equilibrium mixture of at least two species. The redox potential for this mixture was found to be 270 � 10 mV. The major component of this mixture is reduced by A2- with a rate constant of (4.7 � 0.1) × 1081.mol-1 s-1. A value of 747 � 5 mV was measured for the redox potential of the cobalt couple, although equilibration of this system with the inert electrode could be achieved only by using [Fe(bpy)2(CN)2] as a mediator. Kinetics of reduction of [Co(dipic)2]- by ascorbate were complex and not reproducible.

Download Full-text

A study of the kinetics and mechanism of yeast alcohol dehydrogenase with a variety of substrates

Biochemical Journal ◽

10.1042/bj1310261 ◽

1973 ◽

Vol 131 (2) ◽

pp. 261-270 ◽

Cited By ~ 85

Author(s):

F. M. Dickinson ◽

G. P. Monger

Keyword(s):

Alcohol Dehydrogenase ◽

Ethanol Oxidation ◽

Ternary Complexes ◽

Kinetics Of Oxidation ◽

Yeast Alcohol Dehydrogenase ◽

Rate Limiting Step ◽

Limiting Step ◽

Binary Complexes ◽

Rate Limiting ◽

Kinetics Of

1. The kinetics of oxidation of ethanol, propan-1-ol, butan-1-ol and propan-2-ol by NAD+ and of reduction of acetaldehyde and butyraldehyde by NADH catalysed by yeast alcohol dehydrogenase were studied. 2. Results for the aldehyde–NADH reactions are consistent with a compulsory-order mechanism with the rate-limiting step being the dissociation of the product enzyme–NAD+ complex. In contrast the results for the alcohol–NAD+ reactions indicate that some dissociation of coenzyme from the active enzyme–NAD+–alcohol ternary complexes must occur and that the mechanism is not strictly compulsory-order. The rate-limiting step in ethanol oxidation is the dissociation of the product enzyme–NADH complex but with the other alcohols it is probably the catalytic interconversion of ternary complexes. 3. The rate constants describing the combination of NAD+ and NADH with the enzyme and the dissociations of these coenzymes from binary complexes with the enzyme were measured.

Download Full-text