The enzyme aldolase although crystallized in several laboratories in the late 60's proved difficult to solve and it was only 20 years later that our laboratory determined the crystal structure of the first aldolase enzyme in mammals. The research in our laboratory has allowed us to obtain a detailed description as to how aldolase functions at the molecular level. The enzyme catalyzes its chemical reaction with the exquisite simplicity – cleavage of the enzyme substrate, fructose 1,6-bisphosphate, consists of a sequence of chemical reactions that implicate fewer amino acid residues than reaction steps involved. For structural study of molecular events involved in the catalytic cycle by protein crystallography, our approach has been to trap reaction intermediates by first soaking aldolase crystals with reactant for a specific time and then to quench the aldolase crystal to liquid nitrogen temperature. This methodology is perfectly general and has been applied to other crystallized enzymes.

The cryotrapping of reaction intermediates has resulted in snapshots revealing the covalent binding by natural substrate and product and has afforded succinct description of molecular events occurring at the level of the transition state on the enzyme. The chemical reaction catalyzed by aldolase that of cleaving a carbon-carbon bond is also exploited in other cellular metabolic pathways. By applying our cryotrapping technique to crystals of a bacterial aldolase (2-keto-3-deoxy- 6-phosphogluconate aldolase), it was possible to undertake a first ever structural analysis of a genuine covalent enzymatic intermediate, a Schiff base, that is generally very difficult to directly study in enzymes. The structure of the covalent precursor delineated the role of active site residues in this aldolase and the underlying catalytic events.

Generally, different enzymes are used to catalyze different chemical reactions. In case of fructose 1,6-bisphosphate aldolases, we have a rare case of two different enzymes catalyzing the same chemical reaction. By comparing the structure of a bacterial metallo-aldolase and that of the mammalian aldolase, we were able to show that the molecular architecture of the catalytic sites of the mammalian and bacterial aldolases were completely different such that each cleave the same substrate by different catalytic mechanisms. Although mammalian and bacterial aldolases have similar polypeptide folds, active site dissimilarity indicates that evolution of aldolase function arose at a later stage with respect to structure and has made bacterial metallo-aldolases a novel drug target. Cryotrapping of reaction intermediates in several bacterial metallo-aldolases lead us to propose an induced fit mechanism that has provided a comprehensive explanation of the reaction trajectory in these enzymes.