The AdhS alleloenzyme of alcohol dehydrogenase from Drosophila melanogaster. Variation of kinetic parameters with pH.
The variation with pH of the kinetic parameters for the alcohol and acetaldehyde reactions were studied for the alleloenzyme AdhS from Drosophila melanogaster. The variation of Ki (KEO,I) with pH for two ethanol-competitive inhibitors, pyrazole and 2,2,2-trifluoroethanol, was also studied. Both alcohol oxidation and acetaldehyde reduction follow a compulsory ordered pathway, with coenzyme binding first. The rate-limiting step for ethanol oxidation is complex and involves at least hydride transfer and dissociation of the enzyme-NADH complex (ER). In contrast with this, the rate-limiting step for the back reaction, i.e. the reduction of acetaldehyde, is dissociation of the enzyme-NAD+ complex (EO). A rate-limiting ER dissociation appears in the oxidation of the secondary alcohol propan-2-ol, whereas for the back reaction, i.e. acetone reduction, hydride transfer in the ternary complexes is rate-limiting. There is one group in the free enzyme, with a pK of approx. 8.0, that regulates the kon velocity for NADH, whereas for NAD+ several groups seem to be involved. A group in the enzyme is drastically perturbed by the formation of the binary EO complex. Protonation of this group with a pK of 7.6 in the EO complex resulted in weakened alcohol and inhibitor binding, in addition to an increased dissociation rate of NAD+ from the binary EO complex. Neither the binding of acetaldehyde nor the dissociation rate of NADH from the binary ER complex varied within the pH region studied.
Winberg
J O
JO
Biochemical Institute, University of Oslo, Norway.
McKinley-McKee
J S
JS
D000079:Acetaldehyde / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000483:Alleles; D000818:Animals; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D000431:Ethanol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D009243:NAD; D000431:Ethanol; D000426:Alcohol Dehydrogenase; D000079:Acetaldehyde