Article Biochemistry, Vol. 48, No. 43, 2009 10391 FIGURE 2: Kinetics of azide-dependent proteolysis of substrate pG12 by Sbt169. The concentration of the enzyme-substrate complex was 1 μM in 0.1 M KPO4 (pH 7.2, 22 C) and (A) 100 mM azide, (B) 50 mM azide, (C) 10 mM azide, and (D) 1 mM azide. The release of GB is followed by fluorescence for each azide concentration (b). Solid lines are fits to Scheme 1 obtained with KinTek Global Explorer. Similar measurements for formate, cyanate, fluoride, and chloride resulted in the values in Table 1. Reaction rates were measured using a KinTek stopped-flow model SF2001 instrument (excitation λ = 300 nm, and emission with a 450 nm cutoff filter). Stock solutions of Alexafluor-labeled pS169 at a concentration of 100 μM and pG12 at a concentration of 350 μM, both in 100 mM KPi (pH 7.2), were prepared and equilibrated at 22 C. Immediately prior to stop-flow mixing, enzyme and substrate were combined and diluted each to a concentration of 2 μM, in 100 mM KPi (pH 7.2), and placed in syringe A of the SF2001 instrument. Solutions of triggering anion in the same buffer were placed in syringe B. Fluorescence data were collected after 1:1 mixing of the two solutions. The final anion concentration was varied from 0 to 100 mM for azide, from 0 to 500 mM for cyanate and formate, and from 0 to 1 M for fluoride and chloride. The final concentration of the protein complex was fixed at 1 μM. The rates of azide triggering were also determined in 100 mM KPi (pH 7.2) with 250 and 500 mM KCl. The rate of GB release was followed by the decrease in fluorescence at 450 nm. At least 10 kinetic traces were collected for each concentration of anion. Kinetic data were fit using KinTek Global Explorer obtained from KinTek Corp. (www.kintek-corp.com). Crystallization and Structure Analysis. The s164-pG16 complex was crystallized from 15% (v/v) PEG 8000, 0.2 M zinc acetate, and 0.1 M sodium cacodylate (pH 6.2) as previously described (17). For the anion-free complex, a crystal was cryoprotected by immersion for 2 s in 7% glycerol and then cryocooled by immersion in liquid nitrogen. The fluoride-soaked crystal was immersed for 2 min in reservoir solution to which 200 mM KF had been added and then cryoprotected and frozen. The azide complex crystal was immersed for 3 min in reservoir solution to which 25 mM sodium azide had been added, prior to cryoprotection and freezing. Diffraction data for the anion-free crystal were collected on beamline 24ID of the Argonne Synchrotron, while data for the other two crystals were collected on a rotating anode R-axis IV diffractometer. The anion-free structure was determined by molecular replacement as described previously (17). Diffraction data sets to resolutions of 1.7, 1.9, and 1.8 A˚ were collected for the three structures, 3CNQ, 3CO0, and 3BGO, respectively. Refinements of all three structures using REFMAC5 (30) and XFIT (31) led to Rfree values of 0.23, 0.29, and 0.26, respectively. For each of the three structures, a difference map showing the scissile and C-terminal tail regions of the substrate, as well as the side chain of His 64 and the contents of the anion-binding pocket, was calculated before these components had been added to the structure, but after the remainder of the structure had been refined. These key compo- nents were thus built into unbiased maps. The molecular graphics program Coot (32) was used to superpose the three structures. Coordinates and complete statistics are available from the RCSB Protein Data Bank as entries 3CNQ (anion-free), 3CO0 (fluoride-soaked), and 3BGO (azide-bound). RESULTS The kinetics of pS169 activity as a function of azide concen- tration, measured in 0.1 M KPO4 (pH 7.2), are shown in Figure 2.