The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: observation of an enzyme-bound NAD-ketone adduct at 1.4 A resolution by X-ray crystallography.
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 A 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 Calphaatom 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 Kavalue 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 Ka7.6) 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 Kavalue for this residue higher than 10.0.
Benach
J
J
Karolinska Institutet, Novum, Center for Structural Biochemistry, Huddinge, S-14157, Sweden.
Atrian
S
S
Gonzàlez-Duarte
R
R
Ladenstein
R
R
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003196:Computer Graphics; D018360:Crystallography, X-Ray / Q000379:methods; D004330:Drosophila / Q000201:enzymology*; D007659:Ketones / Q000378:metabolism; D016018:Least-Squares Analysis; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D017433:Protein Structure, Secondary
D007659:Ketones; D046911:Macromolecular Substances; D009243:NAD; D000426:Alcohol Dehydrogenase