Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-A resolution

Abstract

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate. A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6. Structure determination to 1.95-A resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues. Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps. Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an epsilon-ammonium salt group. Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues.

Scheme 1

Figure 1

Stereo views of the active site in a native KDPG aldolase protomer. Electron density from a 2-Å annealed omit map encompassing Lys-133 and sulfate ion was contoured at 3σ. Sulfate binding and associated water molecule positions are identical in all subunits. Contacts between non-bonded atoms within hydrogen bonding distance are shown by dotted lines. Lys-133 Nz amine participates in hydrogen bonds with sulfate oxyanions O₁ and O₂, as well as water molecules Wat13 and Wat93; O₁, O₂, and Wat13 accept hydrogen bonds consistent with Lys-133 protonated. The Lys-133 side chain shown is oriented roughly perpendicular to the β-barrel axis and is located near the carboxyl end of the β strands comprising the β-barrel. Drawing was made with the program bobscript (38) and presented by using raster3d (39).

Figure 2

Non-bonded interactions shown in active site of KDPG aldolase. Hydrogen bonds shown by dotted lines correspond to distances between 2.5 and 2.9 Å. Hydrogen bonding by Glu-45 with sulfate oxyanion O₁ requires Glu-45 to be protonated.

Figure 3

Stereoview of the active site of a KDPG aldolase protomer corresponding to native crystals soaked with 10 mM pyruvate and showing electron density from a 1.95-Å annealed omit map contoured at 5σ. The orientation shown in a is similar to the view in Fig. 1. The continuous electron density extending from Lys-133 Nz was interpreted as a pyruvate molecule covalently bound to Lys-133. Electron density in b clearly shows the tetrahedral bonding geometry about pyruvate C₂. View in b corresponds to looking from the pyruvate carboxylate toward Lys-133 Nz, with the plane of the drawing oriented roughly perpendicular with respect to a. The conformation of the carbinolamine intermediate is identical in all subunits. Hydrogen bonding contacts are shown as dotted lines. Hydrogen bonding of pyruvate with Glu-45, Arg-49, and Thr-73 stabilizes the carbinolamine intermediate. Wat35 in subunit A, shown here, is favorably oriented, making hydrogen bonds with both Glu-45 and O₃, to catalyze proton transfers at the level of the carbinolamine intermediate. In the other subunits, Wat35 hydrogen bonds only to Glu-45. Wat35 was also omitted in the annealed difference map calculations, and, because of the smaller O₃ B factor, the difference electron density is slightly elongated toward Wat35 at this contour level.

Scheme 2

Figure 4

Non-bonded interactions in active site of KDPG aldolase in complex with pyruvate. Hydrogen bonds shown by dotted lines correspond to distances between 2.5 and 2.9 Å. Interactions with Wat-35 are subunit dependent; distances to Glu-45 OE2 are 2.8, 3.0, and 3.0 Å and are 2.9, 3.5, and 3.7 Å to pyruvate O₃ in subunits A, B, and C, respectively. Distance between pyruvate C₃ and the center of the ring of Phe-135 is 3.7 Å.