structure, the carboxyamino-pyridinium portion of HI-6 is sandwiched by Tyr124 and Trp286, while the oxime-pyridinium portion is highly mobile and disordered. In the corresponding computed model at the Michaelis-Menten state that was identified from the microsecondscale MMDSs, the mobile oxime group forms a hydrogen-bond network to His447 with the oxime oxygen 5.0-A away from the nonaged sarin phosphorus atom. Our studies suggest a reactivation mechanism of sarinnonaged-mAChE by HI-6 that is outlined as follows: upon binding HI-6 adopts a populated conformation and then converts to a less populated conformation whose oxime oxygen atom has a hydrogen-bond network to His447 that simultaneously forms a hydrogen bond with Glu334 resulting in a tetrad of Glu334-His447-H2O-Oxime; this tetrad not only positions the oxime oxygen atom 5.0-A away from the sarin phosphorus atom but also deprotonates the oxime hydroxyl group; upon deprotonation the oxyanion is less bulky and able to attack the phosphorus atom to form a pentavalent, trigonal Collection, processing, and refinement of diffraction data reader. All assays were performed at 30uC. The data were analyzed using the Enzyme Kinetics module of the Astragalus polysaccharide chemical information program SigmaPlot 9.0. Determination of inhibition constants for oximeNmAChE complexes Inhibition constants of oximeNmAChE complexes were obtained from the apparent Michaelis-Menten constant and the apparent maximal velocity in the presence of HI-6 at various concentrations. The KMapp and Vapp values were determined using the Enzyme Kinetics module of SigmaPlot 9.0. The mode of inhibition was determined by analysis of Lineweaver-Burke plots using the statistical module of SigmaPlot. The best-fit results were obtained by using a mixed partial inhibition model. At least four replicates were measured for each HI-6 concentration. Reactivation kinetics Reactivation kinetics was investigated using the reported discontinuous method. To investigate the reactivation rate under the buffer condition that was similar to the one used during crystal soaking, 20830712 the sarinnonaged-mAChE was incubated with HI-6 at various concentrations in a buffer composed of 32% PEG 750 MME, 100 mM HEPES at pH of 7.0, 25 mM NaCl, at 25uC. After 3, 6, 9, 12, and 15 minutes, samples were withdrawn and the 
activity was measured as described above. Secondary plots of kobs versus the concentration of HI-6 were used to determine the affinity between HI-6 and the phosphonylated enzyme and the rate constant for the displacement of the phosphorous conjugate. The reactivation of diisopropyl fluorophosphate- or 17496168 sarin-inhibited recombinant human AChE by K027 was measured using a reported buffer condition. The use of hAChE was for comparison with literature data. The concentrations of K027 used in the reactivation of the sarinnonagedinhibited hAChE and the DFP-inhibited hAChE were 0.175 mM and 253000 mM, respectively. Site-directed mutagenesis Recombinant ��wild-type��mAChE was used as a template for site-directed mutagenesis introduced using the QuikChange II Site-Directed Mutagenesis Kit. All DNA sequences were verified by sequencing carried out at MWG Biotech. HEK-293F cells were maintained in Dulbecco’s DMEM medium supplemented with 10% fetal calf serum and appropriate antibiotics. Cells were transfected with 25 mg DNA-calcium phosphate co-precipitate. After 24 hours, cells were washed and incubated with fresh media for additional 48 hours. Transfected clones were selected by incubation with med
