The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: observation of an enzyme-bound NAD-ketone adduct at 1.4 Å resolution by X-ray crystallography

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Drosophila alcohol dehydrogenase (DADH) is an NAD +-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones. DADH is the member of the short-chain dehydrogenases/reductases family (SDR) for which the largest amount of biochemical data has been gathered during the last three decades. The crystal structures of one binary form (NAD +) and three ternary complexes with NAD + ·acetone, NAD + ·3-pentanone and NAD + ·cyclohexanone were solved at 2.4, 2.2, 1.4 and 1.6 Å resolution, respectively. From the molecular interactions observed, the reaction mechanism could be inferred. The structure of DADH undergoes a conformational change in order to bind the coenzyme. Furthermore, upon binding of the ketone, a region that was disordered in the apo form (186–191) gets stabilized and closes the active site cavity by creating either a small helix (NAD + ·acetone, NAD + ·3-pentanone) or an ordered loop (NAD + ·cyclohexanone). The active site pocket comprises a hydrophobic bifurcated cavity which explains why the enzyme is more efficient in oxidizing secondary aliphatic alcohols (preferably R form) than primary ones. Difference Fourier maps showed that the ketone inhibitor molecule has undergone a covalent reaction with the coenzyme in all three ternary complexes. Due to the presence of the positively charged ring of the coenzyme (NAD +) and the residue Lys155, the amino acid Tyr151 is in its deprotonated (tyrosinate) state at physiological pH. Tyr151 can subtract a proton from the enolic form of the ketone and catalyze a nucleophilic attack of the C α atom to the C4 position of the coenzyme creating an NAD-ketone adduct. The binding of these NAD-ketone adducts to DADH accounts for the inactivation of the enzyme. The catalytic reaction proceeds in a similar way, involving the same amino acids as in the formation of the NAD-ketone adduct. The p K a value of 9–9.5 obtained by kinetic measurements on apo DADH can be assigned to a protonated Tyr151 which is converted to an unprotonated tyrosinate (p K a 7.6) by the influence of the positively charged nicotinamide ring in the binary enzyme-NAD + form. pH independence during the release of NADH from the binary complex enzyme-NADH can be explained by either a lack of electrostatic interaction between the coenzyme and Tyr151 or an apparent p K a value for this residue higher than 10.0.