the purification protocol: lysis of DNA by DNAse, and MtSK precipitation. The pellet was suspended in 2901691 Tris-HCl 50 mM pH 17855348 7.6 containing KCl 500 mM, and centrifuged. This solution was concentrated down to approximately 20 mL, and 20 mL of Tris-HCl 50 mM pH 7.6 containing KCl 500 mM and 2SO4 2 M was added, M. tuberculosis Shikimate Kinase resulting in a solution containing 1 M 2SO4. This solution was clarified by centrifugation. The supernatant was loaded on a Phenyl Sepharose 16/10 column previously equilibrated with CP 868596 chemical information buffer A 2SO4 1 M, pH 7.6) and the adsorbed material eluted with a linear gradient of buffer B at 1 mL min21. The protein fractions containing the MtSK were pooled and loaded on a Sephacryl S-100 HR column and isocratically eluted with buffer B at 0.25 mL min21. The fractions containing homogeneous recombinant MtSK were pooled, and stored in 85% 2SO4. Protein expression, purification and apparent homogeneity of recombinant MtSK was confirmed by 12% sodium dodecyl sulfatepolyacrylamide gel eletrophoresis stained with Coomassie Brilliant Blue. Protein concentration was determined by the method of Bradford et al. using the Bio-Rad protein assay kit and bovine serum albumin as standard. Oligomeric State Determination The oligomeric state of homogeneous MtSK was determined by size exclusion liquid chromatography on Superdex 200 column. The column was pre-equilibrated with 50 mM Tris-HCl pH 7.6 containing 200 mM NaCl at a flow rate of 0.4 mL min21, with UV detection at 280 nm. The calibration curve was constructed employing the following protein 
standards: ribonuclease A, quimotripsinogen, ovalbumine, albumine, aldolase, catalase, and ferritine. The elution volumes of calibration proteins were used to calculate their corresponding partition coefficient. Blue dextran was utilized for determination of the void volume. Vt is the total bead volume of the column. The Kav values for the standards were plotted versus the logarithm of their corresponding molecular masses. A volume of 100 mL of recombinant protein was loaded on the gel filtration column to obtain Ve for MtSK. Kav ~ Ve {V0 Vt {V0 1 Electrospray Ionization Mass Spectrometry Analysis The subunit molecular mass of recombinant protein preparation was assessed by ESI-MS, employing some adaptations made to the system described by Chassaigne and Lobinski. Samples were analyzed on a triple quadrupole mass spectrometer equipped with a standard electrospray probe, and adjusted to a flow rate of ca. 250 mL min21. The source temperature and needle voltage were maintained constant throughout the experimental data collection, applying a drying gas flow of 200 L h21 and a nebulizer gas flow of 20 L h21. The mass spectrometer was calibrated with intact horse heart myoglobin and its typical cone-voltage induced fragments. The subunit molecular mass of recombinant MtSK was determined by adjusting the mass spectrometer to give a peak with at half-height of 1 mass unit, and the sampling cone-to-skimmer lens voltage controlling the transfer of ions to the mass analyzer was set to 38 V. About 50 pmol of each sample was injected into the electrospray transport solvent. The ESI spectrum was obtained in the multichannel acquisition mode, scanning from 500 to 1,800 m/z at a scan time of 7 s. The mass spectrometer is equipped with MassLynx and Transform software for data acquisition and spectrum handling. Steady-state Kinetics Recombinant MtSK enzyme activity was assayed in the the forward direction b