Millisecond Laue structures of an enzyme-product complex using photocaged substrate analogs.
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.
Stoddard
B L
BL
Division of Basic Sciences, Program in Structural Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. bstoddar@fred.fhcrc.org
Cohen
B E
BE
Brubaker
M
M
Mesecar
A D
AD
Koshland
D E
DE
D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods*; D007521:Isocitrate Dehydrogenase / Q000737:chemistry*; D007523:Isocitrates; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009249:NADP / Q000031:analogs & derivatives; D010782:Photolysis
D007523:Isocitrates; C107652:P(2')-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) nicotinamide adenine dinucleotide phosphate; C098539:caged isocitrate; D009249:NADP; D007521:Isocitrate Dehydrogenase