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

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• 作者：Winberg ; Jan-Olof ; Brendskag ; Michelle Kaaber ; Sylte ; Ingebrigt ; Lindstad ; Rune I. ; McKinley-McKee ; John S.
• 关键词：Drosophila ; short chain alcohol dehydrogenase ; reaction mechanism ; catalytic triad ; active site model ; ADH ; alcohol dehydrogenase (EC 1.1.1.1) ; ADH^S ; slow alleloenzyme from Drosophila melanogaster ; E ; enzyme
• 刊名：Journal of Molecular Biology
• 出版年：1999
• 出版时间：November 26, 1999
• 年：1999
• 卷：294
• 期：2
• 页码：601-616
• 全文大小：334 K

文摘

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD⁺ or the NAD⁺-3-pentanone adduct. 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 ΔH_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 present kinetic study indicates that the role of Lys155 is to lower the pK_a values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD⁺ complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pK_a values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary 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.