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Research elucidates for the first time the structure of the dihydroxyacetone phosphate substrate bound fructose-1,6-bis(phosphate) aldolase Schiff base. The X-ray crystallography to 2.6 Angstrom reveals amino acid residues participating in substrate binding and clarifies the catalytic steps of the enzyme reaction. The structure shows a lysine-glutamine dyad motiff in the enzyme.

Site-directed mutagenesis to substitute lysine-146 with leucine, alanine, histidine or glutamine and to produce mutant protein forms in rabbit muscle aldolase reveals that lysine-146 is essential for the chemical phase of catalysis and C3-C4 bond cleavage and its absence affects both processes. The substrate cleavage rates of the mutant enzymes is less than the standard values and their Schiff base formation rates are also low. The enzymes catalyze the release of carbanion intermediates but affect the rate of C3-C4 bond cleavage, confirming that lysine-146 is the key component of catalysis.

A thermodynamic analysis to study the substrate specificity in fructose-1,6-bisphosphate aldolase is conducted. The results show that the conformational coupling arising during catalysis highly affects the substrate specificity of the compound.

Primary sequence identities based on structural alignment that support the homology and reveal additional mechanistic similarities beyond the common use of a lysine for Schiff-base formation are generated. The kinetic results are consistent with participation of conserved residue or position in the protonation of the carbinolamine intermediate and dehydration of the Schiff base in fructose-1,6-(bis)phosphate aldolase and, by analogy, the class I aldolase family.

The use of transient-state kinetics in single-turnover experiments demonstrates that aldolase utilizes only one anomeric form of fructose-1,6-(bis)phosphate and that the overall rate of Schiff-base formation from this substrate is faster than that of sugar-ring-opening in solution.

The structure of a complex formed by a glycolytic enzyme that catalyzes the cleavage of fructose 1,6-bis(phosphate) and its natural substrate is investigated at 2.3 angstrom. Although the structure of the enzyme is well known, assigning catalytic roles to the various enzymic active-site residues has been hampered by the lack of a structure for the enzyme-substrate complex. Results show that the substrate has one position in the Michaelis complex and another in the covalent complex.