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E. coli (strain BL21) were grown aerobically overnight in LB medium either in a 15 l fermentor (BioFlo 4500, New Brunswick Scientific Company, Edison, NJ, USA) under constant aeration (1 VVM, 37°C) and agitation (400 rpm) or in 1 liter flasks (37°C, 250 rpm). The cells were sedimented (10 min, 10 000 × g), suspended in 500 ml of minimal M9 medium containing 10 mM glucose and incubated for 40 min (37°C, 250 rpm). Bacteria were collected by centrifugation (10 min, 10 000 × g), suspended in 30 ml of 50 mM Tris-HCl buffer, pH 7.4, containing 0.2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.15 M KCl, and frozen at -20°C. After thawing the suspension was sonicated (100 kHz, 3 × 60 sec, on ice), the pellet was removed by centrifugation (30 min, 15 000 × g), and the supernatant was used as a source for enzyme purification.
The extract was placed in a water bath (55°C) under continuous stirring and heated to 50°C. After 5 min, the sample was placed on ice, cooled to 4°C and the precipitate was removed by centrifugation (10 min, 15 000 × g). The supernatant was concentrated to 5.0 ml with Centriplus 10 centrifugal filter units (Amicon Inc., Beverly, MA, USA) and run on a Sephadex G-200 column (Ø 2.4 × 65 cm) calibrated with protein size standards. The chromatography was carried out in 20 mM Tris-HCl buffer, pH 7.4, containing 0.2 mM EDTA and 0.1 M NaCl, at a flow rate of 5 cm . hr-1, and 4-ml fractions were collected. AThTP-synthesizing activity was eluted in two nearly equal peaks, corresponding to molecular masses of 355 ± 14 kDa and 190 ± 4 kDa (n = 2). The first peak was used for subsequent kinetic studies. The purification data are summarized in Table 1. Fig. 1 shows the synthesis of AThTP in the high molecular mass fraction.
As shown in Fig. 2, under standard conditions, AThTP synthesis was not linear with time. Instead, we observed a pronounced lag period in the accumulation of product, allowing no initial rate measurements to be made. The duration of the lag period, τ, as determined by extrapolation of the linear part of the curve to the time axis, was about 1.5 hour.
The influence of hydrogen ion concentration on the enzyme activity was examined at pH values ranging from 5.5 to 9.0. Acetate (50 mM, pH 5.5), maleate (50 mM, pH 6.0–6.5), Tris-maleate (50 mM, pH 7.0), Tris-HCl (50 mM, pH 7.5) and Bis-Tris-propane (50 mM, pH 6.5; 100 mM, pH 6.5–9.0) buffers were used in the assay mixture. As can be seen in Fig. 3, the enzyme has a pH optimum of 6.5 to 7.0 and there is clearly an inhibitory effect of Bis-Tris-propane on the enzyme activity.
The effect of ThDP concentration on the reaction rate was studied within the range of 0.1 to 4 mM at a fixed ADP concentration of 1 mM. The reaction followed Michaelis-Menten kinetics giving a hyperbolic saturation curve (Fig. 4) with an apparent Km value of 4.2 ± 0.5 mM (n = 3) obtained from the direct plot using non-linear regression. Extrapolation from the Hanes plot gave a value of 5.2 mM (Fig. 4, inset).
Fig. 5 illustrates the effect of increasing ADP concentrations on the rate of AThTP synthesis at a fixed concentration of ThDP (0.1 mM). In contrast to the effect of ThDP concentration, varying the ADP concentration did not yield Michealis-Menten kinetics but a sigmoid curve was observed. An S0.5 value of 0.08 mM was estimated, indicating that the apparent affinity of the active sites for ADP is rather high. The nH coefficient calculated from the Hill plot (Fig. 5, inset) was 2.1, in agreement with the possibility of two cooperative binding sites for ADP.
No measurable AThTP synthesis was observed in the absence of divalent metal ions. Among the cations tested (Ca2+, Mg2+ or Mn2+, each at 5 mM), Mn2+ was the most efficient; Mg2+ was 70 % less effective, whereas no measurable AThTP synthesis was observed in the presence of Ca2+. The effect of varying Mg2+ concentrations on the reaction rate was explored at a fixed ThDP concentration of 0.1 mM and ADP concentration of 1 mM. As shown in Fig. 6, the saturation curve was sigmoid when total Mg2+ concentrations were used. This sigmoidicity did not disappear completely when free Mg2+ concentrations (calculated assuming a dissociation constant of 457 μM for Mg- ADP- complex , were used for analysis. The apparent values of S0.5 for total and free Mg2+ were estimated to be about 2.8 mM and 2.0 mM, respectively.
Various combinations of substrates were tested: ADP + ThMP, ADP + ThDP, ADP + ThTP, ATP + ThMP, ATP + ThDP, ATP + ThTP. Among the thiamine phosphates, the enzyme exhibited an absolute specificity for ThDP, whereas both ADP and ATP could serve as the second substrate, the rate of AThTP synthesis being essentially equal with either substrate (data not shown). Our preparation contained no significant ATP hydrolyzing activity, excluding that the activity observed in the presence of ATP was due to its hydrolysis to ADP. No peaks corresponding to a newly synthesized compound were observed on chromatograms when ADP was replaced by GDP, CDP or UDP. There was also no synthesis of compounds such as diadenosine phosphates when a single substrate such as ADP or ATP was used (in this case the reaction was monitored by UV detection after separation on a C18-reversed-phase HPLC column). ThDP alone did not appear to be transformed either.
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