PDBsum entry 3dfs

Lyase

PDB id: 3dfs

Contents

Protein chains

350 a.a. *

Ligands

13P ×4

Waters ×2120

* Residue conservation analysis

PDB id:

3dfs

Name:

Lyase

Title:

Dihydroxyacetone phosphate schiff base intermediate in d33s mutant fructose-1,6-bisphosphate aldolase from rabbit muscle

Structure:

Fructose-bisphosphate aldolase a. Chain: a, b, c, d. Synonym: muscle-type aldolase. Engineered: yes. Mutation: yes

Source:

Oryctolagus cuniculus. Rabbit. Organism_taxid: 9986. Gene: aldoa. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.03Å R-factor: 0.141 R-free: 0.187

Authors:

M.St-Jean,J.Sygusch

Key ref:

M.St-Jean et al. (2009). Charge stabilization and entropy reduction of central lysine residues in fructose-bisphosphate aldolase. Biochemistry, 48, 4528-4537. PubMed id: 19354220

Date:

12-Jun-08 Release date: 28-Apr-09

Protein chains

P00883  (ALDOA_RABIT) -  Fructose-bisphosphate aldolase A from Oryctolagus cuniculus

Seq: 364 a.a.

Struc: 350 a.a.*

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.4.1.2.13 - fructose-bisphosphate aldolase.
• Reaction: beta-D-fructose 1,6-bisphosphate = D-glyceraldehyde 3-phosphate + dihydroxyacetone phosphate

beta-D-fructose 1,6-bisphosphate = D-glyceraldehyde 3-phosphate (Bound ligand (Het Group name = 13P) matches with 90.00% similarity) + dihydroxyacetone phosphate

• Cofactor: Zn(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

Biochemistry 48:4528-4537 (2009)

PubMed id: 19354220

Charge stabilization and entropy reduction of central lysine residues in fructose-bisphosphate aldolase.

M.St-Jean, C.Blonski, J.Sygusch.

ABSTRACT

Fructose-1,6-bisphosphate muscle aldolase is an essential glycolytic enzyme that catalyzes reversible carbon-carbon bond formation by cleaving fructose 1,6-bisphosphate to yield dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde phosphate. To elucidate the mechanistic role of conserved amino acid Asp-33, Asn-33 and Ser-33 mutants were examined by kinetic and structural analyses. The mutations significantly compromised enzymatic activity and carbanion oxidation in presence of DHAP. Detailed structural analysis demonstrated that, like native crystals, Asp-33 mutant crystals, soaked in DHAP solutions, trapped Schiff base-derived intermediates covalently attached to Lys-229. The mutant structures, however, exhibited an abridged conformational change with the helical region (34-65) flanking the active site as well as pK(a) reductions and increased side chain disorder by central lysine residues, Lys-107 and Lys-146. These changes directly affect their interaction with the C-terminal Tyr-363, consistent with the absence of active site binding by the C-terminal region in the presence of phosphate. Lys-146 pK(a) reduction and side chain disorder would further compromise charge stabilization during C-C bond cleavage and proton transfer during enamine formation. These mechanistic impediments explain diminished catalytic activity and a reduced level of carbanion oxidation and are consistent with rate-determining proton transfer observed in the Asn-33 mutant. Asp-33 reduces the entropic cost and augments the enthalpic gain during catalysis by rigidifying Lys-107 and Lys-146, stabilizing their protonated forms, and promoting a conformational change triggered by substrate or obligate product binding, which lower kinetic barriers in C-C bond cleavage and Schiff base-enamine interconversion.