J. Allard et al., Covalent intermediate trapped in 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase structure at 1.95-angstrom resolution, P NAS US, 98(7), 2001, pp. 3679-3684
Citations number
38
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.