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

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 ba se intermediates between Lys-133 and a keto substrate. A covalent adduct wa s trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruva te in acidic conditions at pH 4.6. Structure determination to 1.95-Angstrom 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 r esidues. Carbinolamine interaction with Glu-45 indicates general base catal ysis of several rate steps, Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native struct ure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an E-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.