Theoretical calculations of the catalytic triad in short-chain alcohol dehydrogenases/reductases

Abstract

Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that might be possible when inhibitors bind to the enzyme cofactor complex, were constructed. The binding of the two alcohol competitive inhibitors were studied using automatic docking by the Internal Coordinate Mechanics program, molecular dynamic (MD) simulations with the AMBER program package, calculation of the free energy of ligand binding by the linear interaction energy method, and the hydropathic interactions force field. The calculations indicated that deprotonated Tyr acts as a strong base in the binary enzyme-NAD(+) complex. Molecular dynamic simulations for 5 ns confirmed that deprotonated Tyr is essential for anchoring and orientating the inhibitors at the active site, which might be a general trend for the family of SDRs. The findings here have implications for the development of therapeutically important SDR inhibitors.

FIGURE 1

Ribbon representation of the dimer association of DlADH (PDB ID: 1SBY). Secondary structure nomenclature is as suggested in the publication of the x-ray structure (10) and is shown for one of the subunits. Amino acids lining the R1 and R2 pockets of the active site are shown by the green and blue stick models, respectively. The color coding of secondary structures is red for α-helices, aquamarine for β-strands, magenta for 3–10 helices (γ), and blue for loops. The designation of the active site pockets was followed from the description of crystallographic studies by Benach (11).

SCHEME 1

Compulsory ordered ternary complex mechanism describing the kinetics of DADH catalysis. O is NAD⁺, R is NADH, S is alcohol, and P is aldehyde/ketone. The constants (k and k′) are individual kinetic constants.

FIGURE 2

Mechanism proposed for DADH catalysis. E represents the enzyme, and BH represents an ionizing group with a pK_a value of 7.1 and 7.3 in DlADH at 23.5°C and 19.0°C, respectively (27,71), and 7.6 in DmADH^S (slow alleloenzyme) at 23.5°C (28,30). The insert shows the variation of Φ₂ with varying pH for DlADH at 19.0°C, where Φ₂ = (1/k₂)(1 + (k₋₂/k)(1 + (k′/k′₋₂))) and the kinetic constants are those in Scheme 1. It was shown that only k₂, i.e., the k_on velocity for the alcohol, varied with pH, and hence Φ₂ = Φ*₂ (1 + ([H⁺]/K_a)). The theoretical curve is based on a pK_a of 7.3 and a Φ*₂ of 2.0 mMs.

FIGURE 3

2, 2, 2-trifluoroethanol (TFE) and 1H-PYR.

SCHEME 2

Summary of theoretical calculations. Scenario D is not included in the ranking.

FIGURE 4

Electrostatic surface of the x-ray structure of DlADH. The most electronegative surfaces are in red, whereas the most electropositive areas are in blue. (A) Electrostatic surface of the entire monomeric DlADH. (B) Closer view of the NAD⁺ binding region in the apo form of DlADH active site, viewed as in A. NAD⁺ is shown in a position that corresponds to the position in the binary x-ray crystal structure complex. The NAD⁺ molecule is shown as a stick model and colored according to atom type (carbon-yellow, nitrogen-blue, oxygen-red). A cutting plane has been inserted in the region of the NAD⁺ molecule, and the NAD⁺ bond that is missing in the figure is due to the cutting plane. (C) A closer view of the electrostatic surface of the nicotinamide and ribose binding regions of the active site cavity of the ternary x-ray crystallographic complex DlADH·NAD⁺·TFE (PDB ID: 1SBY). The nicotinamide-binding site is slightly electronegative, whereas the ribose-binding region is strongly electropositive.

FIGURE 5

Ternary DlADH·NAD⁺·PYR complex with the ionization scenario of the catalytic triad after 5 ns of MD. (A) Amino acids lining the R1 (wire model in green) and R2 (wire model in blue) subpockets of the active site. The nicotinamide and ribose ring of NAD⁺, the catalytic triad (Ser-138, Tyr-151, and Lys-155), and PYR are shown as stick models with coloring according to the atom type (carbon-yellow, nitrogen-blue, oxygen-red, and hydrogen-gray). The ribbon represents the C-terminal loop of the other subunit acting as a lid of the active site cavity. PYR is shown both before and after the 5 ns of MD. The ring was parallel to the nicontinamide ring of the NAD⁺ ring at the start of the MD but tilted to perpendicular during the MD. (B) Fluctuation of atomic distances between PYR and atoms of the active site during the MD.

FIGURE 6

Ternary DlADH·NAD⁺·TFE complex with the ionization scenario at the active site. (A) Amino acids lining the R1 (wire model in green) and R2 (wire model in blue) subpockets of the active site. Only selected active site residues are included. The nicotinamide and ribose ring of NAD⁺, catalytic triad (Ser-138, Tyr-151, and Lys-155), and TFE are shown as stick models colored according to the atom type (carbon-yellow, nitrogen-blue, oxygen-red, and hydrogen-gray). (B) Fluctuation of atomic distances between TFE and atoms of the active site during the MD.