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J. Biol. Chem., Vol. 280, Issue 29, 27262-27270, July 22, 2005

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High Resolution Reaction Intermediates of Rabbit Muscle Fructose-1,6-bisphosphate Aldolase

SUBSTRATE CLEAVAGE AND INDUCED FIT^*

Miguel St-Jean, Julien Lafrance-Vanasse, Brigitte Liotard, and Jurgen Sygusch

From the Department of Biochemistry, Université de Montréal, Montréal, Québec H3C 3J7, Canada

Crystal structures were determined to 1.8 Å 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 -helical regions that promote phosphate binding in the active site.

Received for publication, March 3, 2005 , and in revised form, April 20, 2005.

The atomic coordinates and structure factors (codes 1ZAH, 1ZAI, 1ZAJ, and 1ZAL) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by funding from the National Science and Engineering Research Council (Canada) and Canadian Institutes for Health Research. Work was carried out in part at the National Synchrotron Light Source, Brookhaven National Laboratory, which is supported by the United States Dept. of Energy, Division of Materials Sciences and Division of Chemical Sciences under contract DE-AC02-98CH10886. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Biochimie/Médecine, Université de Montréal, CP 6128, Station Centre Ville, Montréal, Quebec H3C 3J7, Canada. Tel.: 514-343-2389; Fax: 514-343-6463; E-mail: Jurgen.Sygusch{at}UMontreal.CA.

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