Carboxy-terminus recruitment induced by substrate binding in eukaryotic fructose bis-phosphate aldolases.
The crystal structures of Leishmania mexicana fructose-1,6-bis(phosphate) aldolase in complex with substrate and competitive inhibitor, mannitol-1,6-bis(phosphate), were solved to 2.2 A resolution. Crystallographic analysis revealed a Schiff base intermediate trapped in the native structure complexed with substrate while the inhibitor was trapped in a conformation mimicking the carbinolamine intermediate. Binding modes corroborated previous structures reported for rabbit muscle aldolase. Amino acid substitution of Gly-312 to Ala, adjacent to the P1-phosphate binding site and unique to trypanosomatids, did not perturb ligand binding in the active site. Ligand attachment ordered amino acid residues 359-367 of the C-terminal region (353-373) that was disordered beyond Asp-358 in the unbound structure, revealing a novel recruitment mechanism of this region by aldolases. C-Terminal peptide ordering is triggered by P1-phosphate binding that induces conformational changes whereby C-terminal Leu-364 contacts P1-phosphate binding residue Arg-313. C-Terminal region capture synergizes additional interactions with subunit surface residues, not perturbed by P1-phosphate binding, and stabilizes C-terminal attachment. Amino acid residues that participate in the capturing interaction are conserved among class I aldolases, indicating a general recruitment mechanism whereby C-terminal capture facilitates active site interactions in subsequent catalytic steps. Recruitment accelerates the enzymatic reaction by using binding energy to reduce configurational entropy during catalysis thereby localizing the conserved C-terminus tyrosine, which mediates proton transfer, proximal to the active site enamine.
Lafrance-Vanasse
Julien
J
Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Sygusch
Jurgen
J
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D007894:Leishmania mexicana / Q000737:chemistry*; D008355:Mannitol Phosphates / Q000737:chemistry; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011817:Rabbits; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D008355:Mannitol Phosphates; D015800:Protozoan Proteins; D005634:Fructose-Bisphosphate Aldolase; D000409:Alanine; D005998:Glycine