The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/r eductase (SDR) family which lack metal ions in their active site. In this f amily, it appears that the three amino acid residues, Ser138, Tyr151 and Ly s155 have a similar function as the catalytic zinc in medium chain dehydrog enases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and tempe rature dependence of various kinetic coefficients of the alcohol dehydrogen ase from Drosophila lebanonensis was studied and three-dimensional models o f the ternary enzyme-coenzyme-substrate complexes were created from the X-r ay crystal coordinates of the D. lebanonensis ADH complexed with either NAD (+) or the NAD(+)-3-pentanone adduct. The k(on) velocity for ethanol and th e ethanol competitive inhibitor pyrazole increased with pH and was regulate d through the ionization of a single group in the binary enzyme-NAD(+) comp lex, with a Delta H-ion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The presen t kinetic study indicates that the role of Lys155 is to lower the pK(a) val ues of both Tyr151 and Ser138 already in the free enzyme. In the binary enz yme-NAD(+) complex, the positive charge of the nicotinamide ring in the coe nzyme further lowers the pK(a) values and generates a strong base in the tw o negatively charged residues Ser138 and Tyr151. With the OH group of an al cohol close to the Ser138 residue, an alcoholate anion is formed in the ter nary enzyme NAD(+) alcohol transition state complex. In the catalytic triad , along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme. (C) 1999 Academic Press.