The structure of a rate-limited product complex formed during a single initial round of turnover by isocitrate dehydrogenase has been determined. Photolytic liberation of either caged substrate or caged cofactor and Laue X-ray data collection were used to visualize the complex, which has a minimum half-life of approximately 10 milliseconds. The experiment was conducted with three different photoreactive compounds, each possessing a unique mechanism leading to the formation of the enzyme–substrate (ES) complex. Photoreaction efficiency and subsequent substrate affinities and binding rates in the crystal are critical parameters for these experiments. The structure suggests that CO2 dissociation is a rapid event that may help drive product formation, and that small conformational changes may contribute to slow product release .
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We thank W. Scott, J. Bolduc, D. Dyer, M. Holmes, R. Strong, and K. Zhang for advice and help with X-ray crystallography, R.M. Sweet, P. Singer, and G. Shea for assistance and technical support at NSLS Beamline X-26C (Brookhaven National Laboratories), D. Ringe and G. Petsko for extended use of their Xe flashlamp, and K. Moffat for extremely helpful advice and criticism at all stages of our time-resolved studies. B.L.S. is funded for this project by the NIH, DEK by the NSF and DOE.
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Stoddard, B., Cohen, B., Brubaker, M. et al. Millisecond Laue structures of an enzyme–product complex using photocaged substrate analogs. Nat Struct Mol Biol 5, 891–897 (1998). https://doi.org/10.1038/2331
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