Drosophila lebanonensis alcohol dehydrogenase:: pH dependence of the kinetic coefficients

The alcohol dehydrogenase (ADH) from Drosophila lebanonensis shows 82% positional identity to the alcohol dehydrogenases from Drosophila melanogaster. These insect ADHs belong to the short-chain dehydrogenase/reductase family which lack metal ions in their active site. In this family, it appears that the function of zinc in medium chain dehydrogenases has been replaced by three amino acids, Ser(138), Tyr(151) and Lys(155). The present work on D. lebanonensis ADH has been performed in order to obtain information about reaction mechanism, and possible differences in topology and electrostatic properties in the vicinity of the catalytic residues in ADHs from various species of Drosophila. Thus the pH dependence of various kinetic coefficients has been studied. Both in the oxidation of alcohols and in the reduction of aldehydes, the reaction mechanism of D. lebanonensis ADH in the pH 6-10 region was consistent with a compulsory ordered pathway, with the coenzymes as the outer substrates. Over the entire pH region, the rate limiting step for the oxidation of secondary alcohols such as propan-2-ol was the release of the coenzyme product from the enzyme-NADH complex. In the oxidation of ethanol at least two steps were rate limiting, the hydride transfer step and the dissociation of NADH from the binary enzyme-NADH product complex. In the reduction of acetaldehyde, the rate limiting step was the dissociation of NAD(+) from the binary enzyme-NAD(+) product complex. The pH dependences of the k(on) velocity curves for the two coenzymes were the opposite of each other, i.e. k(on) increased for NAD(+) and decreased for NADH with increasing pH. The two curves appeared complex and the k(on) velocity for the two coenzymes seemed to be regulated by several groups in the free enzyme. The k(on) velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a pK(a) value of 7.1. The k(on) velocity for acetaldehyde was pH independent and showed that in the enzyme-NADH complex, the pK(a) value of the catalytic residue must be above 10. The k(off) velocity of NAD(+) appeared to be partly regulated by the catalytic residue, and protonation resulted in an increased dissociation rate. The k(off) velocity for NADH and the hydride transfer step was pH independent. In D. lebanonensis ADH, the pK(a) value of the catalytic residue was 0.5 pH units lower than in the ADH(S) alleloenzyme from D. melanogaster. Thus it can be concluded that while most of the topology of the active site is mainly conserved in these two distantly related enzymes, the microenvironment and electrostatic properties around the catalytic residues differ. (C) 1999 Elsevier Science B.V. All rights reserved.