The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: Observation of an enzyme-bound NAD-ketone adduct at 1.4 angstromresolution by X-ray crystallography
J. Benach et al., The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: Observation of an enzyme-bound NAD-ketone adduct at 1.4 angstromresolution by X-ray crystallography, J MOL BIOL, 289(2), 1999, pp. 335-355
Drosophila alcohol dehydrogenase (DADH) is an NAD(+)-dependent enzyme that
catalyzes the oxidation of alcohols to aldehydes/ketones. DADH is the membe
r of the short-chain dehydrogenases/reductases family (SDR) for which the l
argest amount of biochemical data has been gathered during the last three d
ecades. The crystal structures of one binary form (NAD(+)) and three ternar
y complexes with NAD(+). acetone, NAD(+). 3-pentanone and NAD(+). cyclohexa
none were solved at 2.4, 2.2, 1.4 and 1.6 Angstrom 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 th
at 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 po
cket comprises a hydrophobic bifurcated cavity which explains why the enzym
e is more efficient in oxidizing secondary aliphatic alcohols (preferably R
form) than primary ones. Difference Fourier maps showed that the ketone in
hibitor molecule has undergone a covalent reaction with the coenzyme in all
three ternary complexes. Due to the presence of the positively charged rin
g of the coenzyme (NAD(+)) and the residue Lys155, the amino acid Tyr151 is
in its deprotonated (tyrosinate) state at physiological pH. Tyr151 can sub
tract a proton from the enolic form of the ketone and catalyze a nucleophil
ic attack of the C-alpha atom to the C4 position of the coenzyme creating a
n NAD-ketone adduct. The binding of these NAD-ketone adducts to DADH accoun
ts 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 pK(a) value of 9-9.5 obtained by kinetic measurements o
n apo DADH can be assigned to a protonated Tyr151 which is converted to an
unprotonated tyrosinate (pK(a) 7.6) by the influence of the positively char
ged nicotinamide ring in the binary enzyme-NAD(+) form. pH independence dur
ing the release of NADH from the binary complex enzyme-NADH can be explaine
d by either a lack of electrostatic interaction between the coenzyme and Ty
r151 or an apparent pK(a) value for this residue higher than 10.0. (C) 1999
Academic Press.