The ability to identify the interactions formed and broken during productive catalytic turnover is a critical step in the interpretation of reaction mechanisms and the rational design of potentially therapeutic agents which are reactive towards specific biological sites. The time necessary to collect a crystallographic data set for a macromolecule using monochromatic techniques ranges from hours to days. Crystallographers usually examine the interactions formed during catalysis indirectly through the preparation of inhibited or non-productive enzyme-substrate complexes. Therefore, success in producing a correct structural model for enzymatic function based on such studies rests on the investigator's ability to infer how the resulting structure varies from the productive complex of interest. However, it is possible to collect x-ray diffraction data sets with millisecond exposure times by using the high-intensity polychromatic x-ray spectrum available at a synchrotron beamline. Processed data may subsequently be deconvoluted to produce a series of distinct electron density maps corresponding to observable reaction intermediates which accumulate during the duration of the experiment. We will attack a pair of specific aims during the duration of this project, and in doing so will address a more general goal of developing the techniques necessary for kinetic crystallography. 1. We will solve the crystallographic structure of isocitrate dehydrogenase complexed with the catalytic intermediate oxalosuccinate. This enzyme catalyzes the conversion of isocitrate to alpha-ketoglutarate with the liberation of a single carbon dioxide molecule. Catalysis occurs through the transfer of a hydride ion from the alpha-carbon of isocitrate to cofactor, producing a putative oxalosuccinate enol intermediate which decarboxylates through a rate-limiting elimination reaction to form alpha- ketoglutarate. Observation of the intermediate prior to decarboxylation will allow determination of the participants in acid/base catalysis and in local conformational rearrangements caused by the formation of a planar enol group in the substrate molecule. 2. We will solve the crystallographic structure of a pentacoordinate catalytic RNA intermediate prior to self-cleavage. A number of RNA species act as catalysts, and in some cases as true enzymes, including Tetrahymena rRNA L-19 intervening sequence, the RNA subunit of RNAse P, """"""""hammerhead"""""""" domains of plant satellite and virusoid RNAs, and finally, yeast phe-tRNA. This last molecule, which is easily crystallized, undergoes specific, pH- dependent, catalytic cleavage in the presence of various metals, including lead. Thus, this molecule serves as a model for RNA catalysis in more complex systems. We will solve the structure of the relatively stable pentacoordinate intermediate which is formed prior to cleavage, and characterize the interactions formed during turnover and the structural mechanism of RNA self-processing.
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