Glutathionylspermidine synthetase and processes for recovery and use thereof

The present invention describes an enzyme showing glutathionylspermidine synthetase-activity and being distinct from known enzymes with similar activities in several physicochemical parameters, a novel process to isolate said enzyme from Crithidia fasciculata, tools for the production thereof in genetically transformed organisms, and its use as a molecular target for the discovery of trypanocidal drugs.

The standard assay was carried out at 25.0° C. in a volume of 0.15 ml containing 50 mM Bis-Tris-propane, 50 mM Tris, pH 7.5, 5 mM MgSO 4, 1 mM EDTA, 5 mM DTT, 5 mM ATP, 10 mM GSH, 10 mM spermidine, and GspS. After an incubation of 10 min the malachite green reagent was added for detection of GspS activity. High enzymatic GspS activity is visualized by a dark green color of the assay. Blank&equals;standard assay with water instead of GspS; no liberation of phosphate, therefore the color of the assay is yellow instead of green. ADP significantly inhibits GspS. The type of inhibition is competitive with respect to ATP. Concentration of ATP (see heading) and ADP (see table). 1 ATP &lsqb;mM&rsqb;:-> 0.2 mM 0.2 mM 0.5 mM 0.5 mM 1 mM 1 mM 2 mM 2 mM 5 mM 5 mM 1 2 3 4 5 6 7 8 9 10 11 12 A blank 10 10 10 10 10 10 10 10 10 10 B blank 5 5 5 5 5 5 5 5 5 5 C blank 2 2 2 2 2 2 2 2 2 2 D blank 1 1 1 1 1 1 1 1 1 1 E 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 F 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 G 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 H 0 0 0 0 0 0 0 0 0 0 
 EXAMPLES 
 Example 1 
 Purification of GspS Production of starting material: C. fasciculata was grown in a medium previously described (27) in a 100-1 fermenter at 27° C. with continuous stirring (200 rpm) and aeration (0.1 vvm). Organisms were harvested in the late logarithmic growth phase by continous flow centrifugation. The pellet was resuspended with 100 mM HEPES buffer (pH 7.5) containing 1 mM DTT and 1 mM MgSO 4 . After centrifugation the cells were stored at −20° C. GspS, Assay: The assays were carried out at 25.0° C. in a volume of 0.9 ml containing 50 mM Bis-Tris-propane, 50 mM Tris, pH 7.5, 5 mM MgSO 4 , 1 mM EDTA, 5 mM DTT, 5 mM ATP, 10 mM GSH, and 10 mM spermidine (1). The assay for TS was carried out as described by Smith et al. (1). Aliquots were taken after 20 min. For thiol analysis a precolumn derivatization with the fluorescent thiol-specific reagent, monobromobimane (Calbiochem), was used as described previously (2). All samples for HPLC analysis were diluted four-fold with water. Separation and analytical conditions were as described previously (28). HPLC-analysis was performed with a Jasco-HPLC-system consisting of an autosampler (851-AS), a pump (PU-980), a ternary gradient unit (LG-980-02), and a highly sensitive fluorescence detector (FP-920), which enabled a precise analysis of the small product peak in the presence of numerous other and larger ones. An external standard (0.04 mM Gsp) was used for integration calibration of the samples. Extraction in Aqueous Two-Phase Systems: 250 g cells were suspended in 250 ml of 20 mM Bis-Tris-propane puffer, pH 7.5, disrupted by freezing in liquid nitrogen and thawing. The crude homogenate was subjected to an aqueous two-phase extraction at room temperature. All other operations were performed at 4° C. For extraction of GspS aqueous two-phase systems (total mass 900 g) were prepared by weighing in concentrated solutions of the phase components and finally the crude extract ( FIG. 3 ). A poly(ethylene glycol) (PEG)/phosphate system containing 7.5% (w/w) PEG 6000 , 13% (w/w) Na—K-phosphate, pH 7.0, and 40% crude homogenate (or water in the blank systems) was used. The mixture was gently shaken for 10 min at room temperature and separated by centrifugation at 5000 g. The top phase was sucked off and applied to a bottom phase of a blank system. After mixing, centrifugation, and separation of the phases the PEG-rich top phase of the second phase extraction was mixed with a blank bottom phase, adjusted to pH 6.0 with HCl. This third system was mixed again, centrifuged, and separated. Now the GspS was found in the phosphate-rich bottom phase. Diafiltration: The phosphate-rich third bottom phase and other pooled enzyme fractions were diafiltrated with an omega membrane with a cut-off of 30 kDa (Filtron Minisette) using a Pro Flux M12 (Amicon) at 0.2 MPa and a 500-fold volume of 2 mM Bis-Tris-propane buffer, pH 8.0. Resource Q Chromatography: A BioLogic-System (Bio-Rad) was used at 4° C. for all chromatographies. The diafiltrated protein mixture was applied onto a Resource Q column (6 ml) (Pharmacia) equilibrated with 2 mM Bis-Tris-propane buffer, pH 8.0. After washing with 10 column volumes of equilibration buffer, the bound proteins were eluted at a flow rate of 1 ml/min with a gradient of 0.0 to 0.4 M KCl (100% B) as follows: t&equals;0 min, B&equals;0%; t&equals;20 min, B&equals;15%; t&equals;40 min, B&equals;15%; t&equals;60 min, B&equals;30%; t&equals;120 min, B&equals;30%; t&equals;150 min, B&equals;100%. The GspS eluted at 0.27 M KCl and the pooled active fractions were diafiltrated with 2 mM Bis-Tris-propane buffer, pH 6.0. Poros 20 Pi Chromatography: The diafiltrated proteins were applied onto Poros 20 Pi (0.46×10 cm, 1.7 ml) (Perseptive Bioystems) equilibrated with 2 mM Bis-Tris-propane buffer, pH 6.0. After washing with 10 column volumes of equilibration buffer, bound proteins were eluted at a flow rate of 4 ml/min with a gradient of 0 to 1 M NaCl (100% B) as follows: t&equals;0 min, B&equals;0%; t&equals;8 min, B&equals;35%; t&equals;16 min, B&equals;35%; t&equals;17 min, B&equals;37%; t&equals;21 min B &equals;37%; t&equals;25 min, B&equals;100%. GspS eluted at 0.7 M NaCl. Poros 20 PE Chromatography: Pooled active fractions were adjusted to 1 M ammonium sulfate and applied onto a hydrophobic interaction chromatography column Poros 20 PE (0.46×10 cm, 1.7 ml) (Perseptive Biosystems) equilibrated with 20 mM Bis-Tris-propane buffer, pH 8.0, containing 1 M ammonium sulfate, washed with 10 column volumes of equilibration buffer, and eluted with a linear gradient of 1 to 0 M ammonium sulfate and a flow rate of 4 ml/min over 7.5 min. GspS eluted at 0.75 M ammonium sulfate. Pooled active fractions were diafiltrated with 10 mM Bis-Tris-propane buffer, pH 6.8. Mono P Chromatography: The diafiltrated fraction was applied onto a Mono P HR 5/20 column (4 ml) (Pharmacia) for anion exchange chromatography. The column was equilibrated with 10 mM Bis-Tris-propane buffer, pH 6.8. After washing with 10 column volumes of equilibration buffer, bound proteins were eluted with a gradient of 0 to 1 M NaCl (100% B) as follows: t&equals;0 min, B&equals;0%; t&equals;20 min, B&equals;25%; t&equals;40 min, B&equals;25%; t&equals;60 min, B&equals;50%; t&equals;80 min, B&equals;50%; t&equals;100 min, B&equals;100%. The flow rate was 1 ml/min. GspS eluted at 0.45 M NaCl. Results: The purification strategy outlined above resulted in a GspS preparation with a specific activity of 37.6 U/mg at an overall yield of about 20%. The purification factor achieved was 12,500. As is seen from table 1, the phase distribution system applied proved to be highly efficient in enriching GspS. The optimized procedure was based on a factorial design of phase compositions (31), i.e. PEG 6000 /phosphate (7.5/13% (w/w)), PEG 4000 /phosphate (8/14% (w/w)), PEG 1550 /phosphate (9/18% (w/w)), each tested at pH 4.0, 5.5, and 7.0 and containing 40% cell lysate. By centrifugation the cell debris were concentrated in a gum-like interphase if the pH of the system was 5.5. A graphical evaluation of the experimental data (not shown) clearly demonstrated a significant increase in the partition coefficient of GspS with increasing pH, and a decrease in the partition coefficient of the total protein with increasing molecular weight of PEG. The best system, containing 7.5% (w/w) PEG 6000 , 13% (w/w) phosphate, pH 7.0, yielded an extraction of GspS into the top phase ( FIG. 3 ) with a purification factor of 30 in one step. Some residual turbidity left in the top phase of the initial extraction could be eliminated by a second extraction step, mixing the primary top phase with a bottom phase of an identical blank system. By these systems a proteolytic activity, as observed with casein yellow, and an ATPase activity were quantitatively removed by extraction into the bottom phases. Simultaneously GspS was completely separated from trypanothione synthetase (TS) activity. While GspS was recovered completely in the top phase, TS activity was extracted into the bottom phase ( FIG. 3 ), but proved to be unstable and was not purified further. After two a extractions into top phases GspS was essentially free of interfering enzymatic activities and could be precisely quantitated by ATP hydrolysis in the presence of glutathione and spermidine. The final chromatographic purification of GspS, however, was impaired by the high phosphate concentration and viscosity of the top phase in which the enzyme was dissolved. GspS was therefore extracted from the second top phase into the bottom phase of a third system by lowering its pH to 6.0 without loss of activity. The GspS in the phosphate-rich bottom phase was diafiltrated and then could be loaded onto a Resource Q column. GspS thus purified appeared homogeneous by SDS-PAGE ( FIG. 4 ) and by titration curve analysis ( FIG. 5 ). 
 Example 2 
 Determination of Physical Parameters of GspS Molecular mass estimation by gel permeation chromatography: Proteins were applied onto a gel permeation chromatography column Superose 12 (HR 10/30) (Pharmacia) equilibrated with 20 mM Bis-Tris-propane buffer, pH 7.5 containing 0.15 M NaCl and eluted with a flow rate of 0.3 ml/min. Blue dextran (2,000 kDa), thyroglobulin (669 kDa), ferritin (440 kDa), &bgr;-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), and carbonic anhydrase (30 kDa) were used as standards. Gel permeation chromatography on Superose 12 indicated a molecular mass of b 79 l kDa. A small activity peak eluted at about 170 kDa suggesting a slight tendency of the enzyme to dimerize. In essence, however, GspS of C. fasciculata was present as a monomeric enzyme of 79 kDa. Electrophoresis: The subunit molecular weight was determined by SDS-PAGE (28) using a PhastGel Gradient 8-25 (Pharmacia) with the following molecular weight standards: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and &agr;-lactalbumin (14.4 kDa). A subunit molecular mass of GspS of 78 kDa was estimated by SDS-PAGE. The native molecular weight was determined by native PAGE using a PhastGel Gradient 8-25 (Pharmacia) with the same following molecular weight standards and additionally: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and lactate dehydrogenase (140 kDa). A molecular weight of 78 kDa was obtained by gradient gel electrophoresis of the native enzyme. The identity of the 78 kDa band with GspS was confirmed by activity staining, i.e. phosphate liberation upon incubation with Mg 2&plus; -ATP, GSH, and spermidine (not shown). The isoelectric point was determined by isoelectric focusing using a PhastGel IEF 3-9 (Pharmacia) with a broad pI calibration kit and by titration curve analysis with PhastGel IEF 3-9. The latter technique is a two-dimensional electrophoresis. In the first dimension, a pH gradient is generated. The gel is then rotated clockwise 90° and the sample is applied perpendicular to the pH gradient across the middle of the gel (29). For detection of GspS activity after isoelectric focusing the gels were cut into two pieces, one was silver stained for protein detection and the second was incubated for 15 min at room temperature in a solution of 100 mM HEPES, pH 7.0, 5 mM MgSO 4 , 1 mM EDTA, 5 mM DTT, 10 mM GSH, 10 mM spermidine, and 2 mM ATP. After 15 min 2.5 ml of a staining solution containing malachite green, ammonium molybdate and Tween 20 (1) was added. Lanes containing active GspS showed a dark green colour after few minutes. The isoelectric point deduced from isoelectric focusing was at pH 4.6. 
 Example b 3 
 Amino Acid Sequencing SDS-PAGE of purified GspS was performed at a constant current of 20 mA in a separating gel (T&equals;7.5%). For blotting, the proteins were transferred for 1.5 h onto a PVDF-membrane at 40 V/70 mA in a buffer containing 25 mM Tris base, 192 mM glycine, and 10% (v/v) methanol. The blot was stained with Coomassie blue. For peptide sequencing the band corresponding to a molecular mass of 78 kDa was cut out. This material was washed and digested with endoproteinase Lys-C as described before (30) and separated by reversed-phase HPLC (30). Peptide peaks were detected at 214 nm and collected manually. Aliquots of 15-30 &mgr;l were applied directly to biobrene-coated, precycled glass fibre filters of an sequencer (Applied Biosystems 470A) sequencer with standard gas-phase programs of the manufacturer. N-terminal amino acid sequencing proved unsuccessful obviously due to a N-terminal blocking group. After proteolytic cleavage with endoproteinase Lys-C, however, a total of 11 peptides could be recovered from HPLC in a quality to allow sequencing. Out of these peptides, 7 could unambigously be aligned to the deduced GspS sequence of E. coli recently published by Bollinger et al. (25) ( FIG. 1 ). GspS of E. coli and of C. fasciculata thus appeared to be phylogenetically related. But based on the limited sequence information, the sequence similarity between these enzymes, with only 40% identities, appears rather low. 
 Example b 4 
 Kinetic analysis All kinetic experiments were carried out at 25.0° C. in a volume of 0.9 ml containing 50 mM Bis-Tris-propane, 50 mM Tris, pH 7.5, 1 mM EDTA, 5 mM DTT and variable concentrations of ATP (0.10, 0.13, 0.18, 0.28, 0.66 mM ATP), GSH (0.36, 0.47, 0.66, 1.11, 3.57 mM GSH), and spermidine (0.36, 0.47, 0.66, 1.11, 3.57 mM spermidine), respectively. The enzymatic tests for kinetic studies except the ADP inhibition studies were performed in the presence of phosphoenolpyruvate (10 mM) and pyruvate kinase (0.5 units). A fixed magnesium concentration of 5 mM and a GspS content of 0.072 mg (0.923 &mgr;M) was used. Aliquots were taken at 15 min and 30 min. GspS activity was analyzed by product determination as described in example 1. The experimental data thus obtained classify the kinetic mechanism of GspS as an equilibrium random-order mechanism. Whether the complexation of the individual substrates occurs absolutely independently of each other or whether the binding substrates mutually affect affinities of cosubstrates, is less easily decided. The apparent K m values for the different substrates, however, are not significantly affected by the concentrations of the respective cosubstrates. Consequently, the deduced dissociation constants of the corresponding binary, ternary and quarternary complexes are very close for a given substrate and not significantly different. This would indeed imply a mutually independent random addition of substrates. But with regard to the inevitable scatter of data it can not be exluded that some route leading to the quarternary complex is slightly favoured. However, a rapid equilibrium random-order mechanism, as depicted in FIG. 6 , complies best with the experimental data. Based on this assumption, the limiting K m values are defined as the dissociation constants of the quarternary complexes, numerically 0.25±0.02, 2.51±0.33, and 0.47±0.09 mM for Mg 2&plus; -ATP, GSH, and spermidine, respectively. The rate limiting velocity constant then can be calculated to be 415±78 min −1 . A quarternary complex mechanism implies that all three substrates must be assembled at the enzyme before a reaction can proceed. In order to check this hypothesis, we subjected the enzyme to long-term exposure with Mg 2&plus; -ATP plus one of the additional substrates and monitored a potential partial reaction by 31 P NMR. 31 P NMR spectra were recorded on a Bruker ARX 400 NMR spectrometer, at 162 MHz and locked to the deuterium resonance of D 2 O, to detect potential partial reactions. The experiments were carried out at 25.0° C. in a volume of 0.6 ml containing 50 mM Bis-Tris-propane, 50 mM Tris, pH 7.5, 5 mM MgSO 4 , 1 mM EDTA, 5 mM DTT, in the presence of 20% D 2 O. Spectra were recorded at the beginning of the experiment and after the addition of the substrates (5 mM ATP, 10 mM GSH, and 10 mM spermidine). FIG. 8 demonstrates that with all combinations of substrates no ATP turnover could be observed within 5 hours unless the third substrate was added. These findings strongly support the assumption of a quarternary complex mechanism and explain the absence of any ATPase activity of GspS. Neither can the presumed catalytic intermediate glutathionylphosphate be formed in any detectable amount by an incomplete catalytic complex. As already mentioned ADP significantly inhibits GspS which renders it difficult to measure GspS activity in the absence of an ATP regenerating system. The type of inhibition is competitive with respect to ATP. A K i of 80 &mgr; M was calculated which is in the range of physiological ADP concentrations. GspS also proved to be feed-back inhibited by TSH with a K i of 480 &mgr;M, which is competed by GSH. 
 Example b 5 
 pH-Optimum of GspS The activity of GspS was measured essentially as described in example 4 but at pH values ranging from 6 -8. GspS shows a flat pH optimum near pH 7.5 ( FIG. 7 ). 
 Example 6 
 Use of Malachite Green Colorimetric Assay for Liberation of Inorganic Phosphate in GspS Preparations Partially Purified According to Example b 1 The liberation of inorganic phosphate from ATP can be easily visualized by malachite green ( FIG. 9 ). The test is amply used to monitor ATP hydrolyzing activities and is correspondingly unspecific. Surprisingly, the GspS preparation after aqueous two phase extraction, as described in example 1, proved to be free of any significant spontaneous ATP-hydrolyzing activity. This finding enabled us to use this fast and convenient test to measure specifically GspS activity which is accompanied by release of inorganic phosphate from ATP in the presence of glutathione, spermidine, and magnesium ions. The test can be easily adapted to mass screening as outlined below. The malachite green calorimetric assay for liberation of inorganic phosphate (1l) was used for fast detection of GspS activity during purification after column chromatography and for GspS localization on gels. The disclosure comprises also that of EP 96 120 014.4 filed Dec. 12, 1996, the entire disclosure of which is incorporated herein by reference. 
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