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

Abstract

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.

Estimation of rate dissociation constants involving ternary complexes in reactions catalysed by yeast alcohol dehydrogenase

“…1 also suggest that the absorption coefficient for NADH in the complex might be slightly decreased, but are not sufficiently detailed to establish the point. Since the absorption coefficient of the bound NADH is similar to that of free NADH at 340nm (Dickenson, 1975), the results of Fig. I indicate that the enzyme-bound NADH has a fluorescence roughly equivalent to that of free NADH under our conditions.…”

“…(4) The procedure used by Dickenson & Dickinson (1975a) to obtain k43 also provides a value for k'/k'3 (Dickenson, 1975), from which an estimate of k'3 can be obtained. The value of K3 calculated from k ' 3/k43 may be compared with the value in Table 1. (5) The reciprocal maximum rate of isotope exchange (NAD+ = NADH) at equilibrium at pH 7.9 (Silverstein &Boyer, 1964) is, since k/k4> 1, given by:…”

Revealing Ligand Binding Sites and Quantifying Subunit Variants of Noncovalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment

“…It is interesting to note that each yADH subunit binds to two zinc atoms, but no NAD + binding was observed. This might reflect the fact that yADH has lower binding affinity for NAD + than hADH does, differing by up to 1 or 2 orders of magnitude at pH 8 [53, 54]. …”

Displacement of equilibrium in electroenzymatic reactor for acetaldehyde production using yeast alcohol dehydrogenase

“…The routine written to solve Eqs. (8) and (9) and to calculate the catalytic current was used as a subroutine in a nonlinear regression program using Marquardt's algorithm. 16 The curves in Figures 2 and 3 were calculated using the identified parameters.…”