High resolution reaction intermediates of rabbit muscle fructose-1,6-bisphosphate aldolase: substrate cleavage and induced fit

• Published in: The Journal of biological chemistry 2005-07, Vol.280 (29), p.27262-27270
• Main Authors: St-Jean, Miguel, Lafrance-Vanasse, Julien, Liotard, Brigitte, Sygusch, Jurgen
• Format: Article
• Language: English
• Subjects: 08 HYDROGEN; ALDOLASES; Animals; Binding Sites; Catalysis; CLEAVAGE; CONFORMATIONAL CHANGES; CRYSTAL STRUCTURE; Crystallography, X-Ray; Fructose-Bisphosphate Aldolase - chemistry; Fructose-Bisphosphate Aldolase - metabolism; Fructosediphosphates - chemistry; Fructosediphosphates - metabolism; HYDROGEN; Hydrogen Bonding; Mannitol Phosphates - chemistry; MATERIALS SCIENCE; Muscle, Skeletal - enzymology; MUSCLES; national synchrotron light source; Protein Conformation; RABBITS; REACTION INTERMEDIATES; RESOLUTION; SCHIFF BASES; Stereoisomerism; Substrate Specificity; SUBSTRATES
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M502413200

Crystal structures were determined to 1.8 A resolution of the glycolytic enzyme fructose-1,6-bis(phosphate) aldolase trapped in complex with its substrate and a competitive inhibitor, mannitol-1,6-bis(phosphate). The enzyme substrate complex corresponded to the postulated Schiff base intermediate and has reaction geometry consistent with incipient C3-C4 bond cleavage catalyzed Glu-187, which is adjacent by to the Schiff base forming Lys-229. Atom arrangement about the cleaved bond in the reaction intermediate mimics a pericyclic transition state occurring in nonenzymatic aldol condensations. Lys-146 hydrogen-bonds the substrate C4 hydroxyl and assists substrate cleavage by stabilizing the developing negative charge on the C4 hydroxyl during proton abstraction. Mannitol-1,6-bis(phosphate) forms a noncovalent complex in the active site whose binding geometry mimics the covalent carbinolamine precursor. Glu-187 hydrogen-bonds the C2 hydroxyl of the inhibitor in the enzyme complex, substantiating a proton transfer role by Glu-187 in catalyzing the conversion of the carbinolamine intermediate to Schiff base. Modeling of the acyclic substrate configuration into the active site shows Glu-187, in acid form, hydrogen-bonding both substrate C2 carbonyl and C4 hydroxyl, thereby aligning the substrate ketose for nucleophilic attack by Lys-229. The multifunctional role of Glu-187 epitomizes a canonical mechanistic feature conserved in Schiff base-forming aldolases catalyzing carbohydrate metabolism. Trapping of tagatose-1,6-bis(phosphate), a diastereoisomer of fructose 1,6-bis(phosphate), displayed stereospecific discrimination and reduced ketohexose binding specificity. Each ligand induces homologous conformational changes in two adjacent alpha-helical regions that promote phosphate binding in the active site.