PDBsum entry 3b8d

Lyase

PDB id: 3b8d

Contents

Protein chains

363 a.a.

Ligands

SO4 ×2

Waters ×3452

References listed in PDB file

Key reference

Title

A conserved glutamate residue exhibits multifunctional catalytic roles in d-Fructose-1,6-Bisphosphate aldolases.

Authors

A.Maurady, A.Zdanov, D.De moissac, D.Beaudry, J.Sygusch.

Ref.

J Biol Chem, 2002, 277, 9474-9483. [DOI no: 10.1074/jbc.M107600200]

PubMed id

11779856

Abstract

The aldolase catalytic cycle consists of a number of proton transfers that interconvert covalent enzyme intermediates. Glu-187 is a conserved amino acid that is located in the mammalian fructose-1,6-bisphosphate aldolase active site. Its central location, within hydrogen bonding distance of three other conserved active site residues: Lys-146, Glu-189, and Schiff base-forming Lys-229, makes it an ideal candidate for mediating proton transfers. Point mutations, Glu-187--> Gln, Ala, which would inhibit proton transfers significantly, compromise activity. Trapping of enzymatic intermediates in Glu-187 mutants defines a proton transfer role for Glu-187 in substrate cleavage and Schiff base formation. Structural data show that loss of Glu-187 negative charge results in hydrogen bond formation between Lys-146 and Lys-229 consistent with a basic pK(a) for Lys-229 in native enzyme and supporting nucleophilic activation of Lys-229 by Glu-187 during Schiff base formation. The crystal structures also substantiate Glu-187 and Glu-189 as present in ionized form in native enzyme, compatible with their role of catalyzing proton exchange with solvent as indicated from solvent isotope effects. The proton exchange mechanism ensures Glu-187 basicity throughout the catalytic cycle requisite for mediating proton transfer and electrostatic stabilization of ketamine intermediates. Glutamate general base catalysis is a recurrent evolutionary feature of Schiff base0forming aldolases.

Figure 3.
Fig. 3. Stereoview of electron density showing Gln-187, Glu-189, and Arg-148 residues in the active site of the E187Q mutant structure. The mutant structure is shown superimposed with equivalent residues in the native enzyme (dark green). Gln-187 donates a hydrogen bond to Glu-189 in E187Q whereas Arg-148 makes additional hydrogen bonds with Glu-189 in E187Q not observed in the native structure. Wat-1376 makes a hydrogen bond to Glu-189 whereas Wat-1647 interacts with Glu-189 and Wat-1856. Electron density shown correspond to a 2F[o] F[c] omit map of residue Gln-187 and contoured at the 1 level.

Figure 4.
Fig. 4. Stereoview of electron density showing superposition of Lys-146, Gln-187, Lys-229, and Leu-270 in E187Q mutant with equivalent residues in the native enzyme (dark green). The hydrogen bond between lysine residues requires that one lysine residue acts as hydrogen bond acceptor. Glu-187 in the native structure is situated within hydrogen bonding distance between the two lysine residues. Wat-8272 makes hydrogen bonds to Lys-146 and Wat-8338 whereas Leu-270 makes close contact with Lys-229. Electron densities shown correspond to a 2F[o] F[c] omit map of residues Lys-146 and Lys-229 and contoured at the 1 level.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2002, 277, 9474-9483) copyright 2002.