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The relative conformational stability of the alcohol dehydrogenase alleloenzymes of the fruitfly Drosophila melanogaster.

ABSTRACT: The effect of temperature on four purified alleloenzymes of the alcohol dehydrogenase (Adhs, Adhf, AdhD and Adhn-5) of the fruitfly Drosophila melanogaster was investigated in detail. Initial-velocity studies showed that the naturally occurring Adhf and Adhs enzymes differed only in their temperature optima, and evidence of kinetic adaptation to high and low temperature was not apparent. All four alleloenzymes denatured irreversibly on heating purified enzyme solutions at pH 6.0. This technique revealed only small differences in thermostability between Adhf and Adhs, although the two mutant enzymes from AdhD and Adhn-5 were considerably more labile. Electrophoresis of the enzymes though a stable transverse temperature gradient proved to be a discriminating and reproducible technique. Enzymes of different net charge were compared on the same polyacrylamide gel. The Adhf enzyme was shown to be significantly less stable than the Adhs enzyme. Subunit interchange was observed at temperatures below the point at which the unfolding occurred. At pH 4.0, the Adhf/Adhs heterodimer was as stable as the Adhs homodimeric enzyme, and more stable than the Adhf homodimer. Adhn-5 and AdhD alleloenzymes were relatively thermolabile. The stability of the alleloenzymes towards urea denaturation was studied by urea-gradient electrophoresis. Only small differences in stability between the Adhf and Adhs enzymes were observed. The AdhD and Adhn-5 mutants were denatured at the same urea concentration, which was much lower than in the case of the wild-type enzymes. Except at pH 4.0, subunit dissociation could not be distinguished from the unfolding of the monomer.

SUBMITTER: Thatcher DR

PROVIDER: S-EPMC1163060 | biostudies-other | 1981 Jul

REPOSITORIES: biostudies-other

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Project description:Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain reliable results for the liberation of NADH during the initial-rate phase, the reaction was measured with a sensitive recording filter fluorimeter, and the complexes formed with the different dead-end and product inhibitors have been interpreted on the basis of a full dismutation reaction. The results are only consistent with a compulsory ordered reaction mechanism, with the formation of a dead-end binary enzyme-aldehyde complex. Under initial-velocity conditions, the rate of acetate release was calculated to be larger than 2.5 s-1, which is more than ten times that of NADH. The substrate specificity constant (kcat/Km or 1/phiB) with respect to the oxidation of substrates was propan-2-ol>ethanol>acetaldehyde>trimethylacetaldehyde.

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The relative conformational stability of the alcohol dehydrogenase alleloenzymes of the fruitfly Drosophila melanogaster.

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