The reactions of 1,10-phenanthroline with yeast alcohol dehydrogenase

Freshly prepared samples of yeast alcohol dehydrogenase (EC 1.1.1.1) were inhibited by 1,10-phenanthroline at pH 7.0 and 0 degrees C in a two-stage process. The first step appeared to be slowly established, but was rendered reversible by removal of reagent or by addition of excess Zn2+ ions. The second step was irreversible and was associated with the dissociation of the tetrameric enzyme. The presence of saturating concentrations of NAD+ or NADH promoted and enhanced inhibition by the slowly established reversible process, but prevented dissociation of the enzyme. For the incubation mixtures containing NAD+, removal of the 1,10-phenanthroline resulted in virtually complete recovery of activity, whereas, for the incubation mixtures containing NADH, removal of the reagent gave only partial re-activation. The presence of NAD+ and pyrazole, or NADH and acetamide, in incubation mixtures with the enzyme gave rise to ternary complexes that gave protection against both forms of inactivation by 1,10-phenanthroline. The results support the view that at least some of the Zn2+ ions associated with yeast alcohol dehydrogenase have a catalytic, as opposed to a purely structural, role.

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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.

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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.

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Role of the essential thiol groups of yeast alcohol dehydrogenase

Biochemical Journal ◽

10.1042/bj1260133 ◽

1972 ◽

Vol 126 (1) ◽

pp. 133-138 ◽

Cited By ~ 19

Author(s):

F. M. Dickinson

Keyword(s):

Alcohol Dehydrogenase ◽

Dissociation Constant ◽

Binding Capacity ◽

Ternary Complexes ◽

Native Enzyme ◽

Thiol Groups ◽

Yeast Alcohol Dehydrogenase

1. Yeast alcohol dehydrogenase inactivated by reaction with iodoacetamide retains 85% of the original NADH-binding capacity as measured under conditions of saturating coenzyme concentration. 2. The dissociation constant of the enzyme–NADH complex is unaffected by inactivation of the enzyme with iodoacetamide, and the affinity of the enzyme for NAD+ and pyridine-3-aldehyde–adenine dinucleotide (PAAD+) appears to be similarly unaffected. 3. Enzyme inactivated with iodoacetamide has lost the ability to form normal ternary complexes of the type enzyme–NADH–acetamide and enzyme–PAAD+–hydroxylamine that are characteristic of the native enzyme.

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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.

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Carboxyl groups near the active site zinc contribute to catalysis in yeast alcohol dehydrogenase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)60737-9 ◽

1988 ◽

Vol 263 (11) ◽

pp. 5446-5454

Author(s):

A J Ganzhorn ◽

B V Plapp

Keyword(s):

Alcohol Dehydrogenase ◽

Active Site ◽

Carboxyl Groups ◽

Yeast Alcohol Dehydrogenase

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Two-Stage Biodiesel Synthesis from Used Cooking Oil with a High Acid Value via an Ultrasound-Assisted Method

Energies ◽

10.3390/en14123703 ◽

2021 ◽

Vol 14 (12) ◽

pp. 3703

Author(s):

Ming-Chien Hsiao ◽

Wei-Ting Lin ◽

Wei-Cheng Chiu ◽

Shuhn-Shyurng Hou

Keyword(s):

Acid Value ◽

Molar Ratio ◽

Optimal Conditions ◽

Two Stage ◽

Cooking Oil ◽

High Acid ◽

Used Cooking Oil ◽

Biodiesel Synthesis ◽

Stage Process ◽

Ultrasound Assisted

In this study, ultrasound was used to accelerate two-stage (esterification–transesterification) catalytic synthesis of biodiesel from used cooking oil, which originally had a high acid value (4.35 mg KOH/g). In the first stage, acid-catalyzed esterification reaction conditions were developed with a 9:1 methanol/oil molar ratio, sulfuric acid dosage at 2 wt %, and a reaction temperature of 60 °C. Under ultrasound irradiation for 40 min, the acid value was effectively decreased from 4.35 to 1.67 mg KOH/g, which was decreased to a sufficient level (<2 mg KOH/g) to avoid the saponification problem for the subsequent transesterification reaction. In the following stage, base-catalyzed transesterification reactions were carried out with a 12:1 methanol/oil molar ratio, a sodium hydroxide dosage of 1 wt %, and a reaction temperature of 65 °C. Under ultrasound-assisted transesterification for 40 min, the conversion rate of biodiesel reached 97.05%, which met the requirement of EN 14214 standard, i.e., 96.5% minimum. In order to evaluate and explore the improvement of the ultrasound-assisted two-stage (esterification–transesterification) process in shortening the reaction time, additional two-stage biodiesel synthesis experiments using the traditional mechanical stirring method under the optimal conditions were further carried out in this study. It was found that, under the same optimal conditions, using the ultrasound-assisted two-stage process, the total reaction time was significantly reduced to only 80 min, which was much shorter than the total time required by the conventional method of 140 min. It is worth noting that compared with the traditional method without ultrasound, the intensification of the ultrasound-assisted two-stage process significantly shortened the total time from 140 min to 80 min, which is a reduction of 42.9%. It was concluded that the ultrasound-assisted two-stage (esterification–transesterification) catalytic process is an effective and time-saving method for synthesizing biodiesel from used cooking oil with a high acid value.

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