Kinetic effect of some aliphatic amines on yeast alcohol dehydrogenase

1976 ◽  
Vol 54 (5) ◽  
pp. 432-437
Author(s):  
M. J. Dove ◽  
C. S. Tsai

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.

1975 ◽  
Vol 147 (2) ◽  
pp. 303-311 ◽  
Author(s):  
C J Dickenson ◽  
F M Dickinson

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.


1977 ◽  
Vol 161 (1) ◽  
pp. 73-82 ◽  
Author(s):  
C J Dickenson ◽  
F M Dickinson

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.


1975 ◽  
Vol 147 (3) ◽  
pp. 541-547 ◽  
Author(s):  
C J Dickenson ◽  
F M Dickinson

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.


1973 ◽  
Vol 131 (2) ◽  
pp. 261-270 ◽  
Author(s):  
F. M. Dickinson ◽  
G. P. Monger

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.


1978 ◽  
Vol 253 (23) ◽  
pp. 8414-8419
Author(s):  
H. Jörnvall ◽  
H. Eklund ◽  
C.I. Brändén

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