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

Abstract

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 C 3 -C 4 bond cleavage catalyzed by Glu-187, which is adjacent 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 C 4 hydroxyl and assists substrate cleavage by stabilizing the developing negative charge on the C 4 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 C 2 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 C 2 carbonyl and C 4 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.Aldolases are ubiquitous enzymes and have been a subject of continuous interest because of their ability to catalyze carboncarbon bond formation in living organisms. Their role is best known in glycolysis, where fructose 1,6-bis(phosphate) (FBP) 1 aldolases (EC 4.1.2.13) promote the reversible cleavage of FBP to triose phosphates, D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). The class I enzyme uses covalent catalysis, implicating a Schiff base formed between a lysine residue on the enzyme and a ketose substrate. In vertebrates, there are three tissue-specific class I aldolases (aldolase A (found in skeletal muscle and red blood cells), aldolase B (found in liver, kidney, and small intestine), and aldolase C (found in neuronal tissues and smooth muscle)), and they are distinguishable on the basis of immunological and kinetic properties (1). The catalytic mechanism has been extensively studied using class I aldolase A from rabbit muscle, and key intermediates are depicted in Scheme I.In the forward reaction, a reactive lysine residue in the active site attacks the ketose (2) of the acyclic FBP substrate (3, 4). Transient formation of a dipolar tetrahedral carbinolamine with the keto function yi...

Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

“…Aldolase concentration was determined using an extinction coefficient of 0.91 (mg/ ml) Ϫ1 at 280 nm (20). WT and K146M aldolases were crystallized using the conditions reported previously (15).…”

“…Purification and Crystallization-Expression and purification of recombinant native (WT) and Lys 146 3 Met mutant (K146M) rabbit muscle aldolases were performed as described previously (11,15,19) and using Escherichia coli strain BL21 SI for overexpression of recombinant proteins (Invitrogen). Aldolase concentration was determined using an extinction coefficient of 0.91 (mg/ ml) Ϫ1 at 280 nm (20).…”

“…The interconversion between the two forms implicates the conserved C-terminal Tyr 363 residue whose proteolysis inhibits the stereospecific proton exchange step, making it rate-limiting (10), whereas the penultimate residues (357-362) of the C-terminal region modulate the rate of exchange reaction (11). The C-terminal region (residues 343-363) is conformationally mobile (12, 13), has an extended secondary structure (14,15), and distinguishes mammalian aldo-* This work was supported in part by the Natural 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 Department of Energy, Division of Materials Sciences and Division of Chemical Sciences under contract DE-AC02-98CH10886.…”

“…The C-terminal region (residues 343-363) is conformationally mobile (12,13), has an extended secondary structure (14,15), and distinguishes mammalian aldo-lases and their orthologues. Although a number of structural studies have been performed that have investigated intermediates in class I aldolases (15)(16)(17)(18), characterization of the interconversion between the iminium and the enamine has so far proven elusive at the structural level.…”

Structure of fructose bisphosphate aldolase fromEncephalitozoon cuniculi

“…Evidence that the monomeric and dimeric forms of EcFBPA interconvert was found in the sizeexclusion chromatogram, which contains a small peak for the dimer and a larger peak for the monomer. Dimers are found in both of the EcFBPA structures presented here; the computed buried surface area at the dimer interface is $3000 Å 2 for both structures, while that for the tetramer interface of rabbit muscle FBPA A (PDB entry 1zai; St-Jean et al, 2005) is $13 500 Å 2 , with each dimer interface burying $4000 Å 2 (Krissinel & Henrick, 2007). Figs.…”