Structural investigation of reaction intermediates in glycolytic aldolases

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A wealth of information has been obtained about catalysis and thermodynamics of enzyme reactions. However, much less is known about the kinetics and energetics of conformational processes in the enzyme. How enzymes progress their reaction intermediates through the catalytic cycle remains an open question in enzymology. The challenge is to unravel the intimate linkage between protein motion and enzymatic function. Protein crystallography conducted at room temperature (RT) is a tool that can be used to analyze conformational motions of enzymes. We will analyze the electron density obtained from crystallographic studies in terms of one or more conformational states for each amino acid (aa) in the protein sequence. If one aa conformation overlaps with a neighboring aa conformation, it can represent a conduit for transmitting conformational motion in the enzyme. These networks can extend across the entire enzyme and correspond to dynamics-driven allostery that does not change the equilibrium positions of atoms. The goal will be to discern which contact networks interact with the enzyme reaction intermediates to understand how these interactions facilitate the chemical transformation of one intermediate into another. We have extensively investigated the glycolytic enzyme aldolase to unravel active site interactions with reaction intermediates and now wish to understand how conformational motions progress the reaction intermediates through the catalytic cycle. Aldolases are enzymes responsible for the generation of stored biological energy and metabolic precursors. We will approach this objective experimentally by investigating contact networks using RT protein crystallography in aldolases, inhibited or inactivated distant from the active site. Would interruption/loss of contact networks explain these allosteric blockages? Can appropriate mutations rescue activity by re-establishing appropriate contact networks? To assess changes in contact networks due to active site events, aldolase mutants will be used that populate consecutive intermediates in the active site. Would changes be confined to a single contact network that reorganizes locally as a function of the reaction intermediate, or are there large scale changes in contact networks for each intermediate, extending to even subunit interfaces? Finally, we will implement a time-resolved crystallographic method using a highly intense micron sized X-ray beam at the NSLS-II synchrotron to collect data from microcrystals over the course of an enzyme reaction. The information captured enables a complete description of the conformational motions during catalysis including actual chemical bond breakage taking place, never seen before on an enzyme! Numerous applications are possible in drug design, biosensors, biomimetics; and offers fundamental insight into cellular processes.