CONCLUSIÓN DEL ANÁLISIS DE LA COMPARACIÓN ENTRE LOS DOCENTES PARA

regulators

With the stably phosphorylated CheF mutant, the phosphotransfer reaction from the kinase to the response regulators can be explored (Fig. 33).

Fig. 33. Transfer of phosphate groups from CheF:D729K-phosphate to CheY and CheV2. A. CheF:D729K-Pi (2.9 µM) was incubated with an equal amount of either CheY

or CheV2 in 5 mM MgCl2, 50 mM potassium phosphate pH 7.5 at room temperature.

Samples (20 µl) were taken from the reaction mixture, immediately mixed at the time points indicated (above autoradiograph) with an equal volume of 2x SDS-buffer and frozen in liquid nitrogen. After thawing, the proteins were separated by SDS-PAGE and transferred to a PVDF membrane. Exposure to X-ray film was overnight. B. The same experiment as described in A with the difference that the response regulator concentration was one tenth of the CheF:D729K-Pi concentration (0.3 and 2.9 µM,

respectively). Phosphorylated proteins are indicated by arrows.

Phosphorylated CheF:D729K transfers its phosphate group very rapidly to the response regulator CheY (within the first ten sec of the experiment). Thereafter, the intensity of the CheF:D729K band does not change significantly, and residual label bound to the kinase is no longer transferred to the response regulator. This is in accordance with the time frame expected from data derived from the E. coli CheA to CheY phosphotransfer reaction (50 ms as measured in a stopped flow apparatus; STEWART, 1997), indicating a role for H. pylori CheY similar to

the one of E. coli. In analogy to E. coli CheY, the life-time of H. pylori CheY-phosphate is short: the band intensity of CheY-phosphate is

decreased to approximately 50 % in lane 3 compared to lane 2 in Fig. 33, indicating an approximate life-time of CheY-phosphate of around 10 sec. Since CheF:D729K-Pi is stable without CheY (see 35, p. 66), the loss of

phosphate groups must be due to CheY autodephosphorylation, and so it appears that CheY catalyzes its own dephosphorylation efficiently, since acyl phosphates have an expected half-life of several hours at neutral pH without catalysis (HESS et al., 1988; STOCK et al., 1995).

In contrast to CheY, CheV2 behaves very different. It also accepts phosphate groups from CheF:D729K-Pi, yet this reaction is slow. From the

CheV2 band intensities it follows that the CheV2-phosphate concentration is always lower as the CheY-phosphate concentration in the analogous reaction. Furthermore, the reaction proceeds considerably longer as with CheY. Even 60 s after mixing the kinase with the response regulator, most of the label is still bound to the kinase, and the CheF:D729K-Pi band is not

yet reduced to 50 % as compared to the band at 0 s. The transfer reaction that is completed within 50 ms in the case of E. coli CheA/CheY takes more than one minute with H. pylori CheV2. This might be due to a slow transfer of label from the kinase to the response regulator. Alternatively, CheV2 might not be phosphorylated to the same extent like CheY. In an extreme case, only a small fraction of the CheV2 proteins might accept phosphate groups from CheF (high portion of inactive CheV2 in the preparation, for example). The transfer reaction could than be still rapid, but the hydrolysis of CheV2-phosphate would become rate-limiting.

The CheV2-phosphate concentration appears to be constant during the first 60 s of the experiment (Fig. 33A), and therefore the half-life of CheV2-phosphate equals the half-life of CheF:D729K-Pi hydrolysis

(hydrolysis of CheV2 becomes time dependent for the reaction). It might be estimated from Fig. 33 to be > 1 min. When the phosphorylated kinase

was used in a tenfold molar excess to the response regulators as in Fig. 33B, the differences in CheY and CheV2 behavior became again apparent: CheY rapidly accepts and hydrolyses all phosphate groups from CheF:D729K, whereas the reaction with CheV2 is considerably reduced. To quantify the amount of phosphate that is transferred from CheF:D729K-Pi

to the respective response regulators, identical experiments as the one described in Fig. 33 where performed with the modification that the CheF bands were excised from the gel and the radioactivity bound to the protein was determined by liquid scintillation counting (Fig. 34).

Fig. 34. Loss of label from CheF:D729K-Pi to CheY. Phosphorylated CheF:D729K (4

µM) was incubated at 25°C with various concentrations of the response regulators. At the time intervals indicated, 20 µl samples were taken, immediately mixed with an equal volume of 2x SDS-buffer and frozen in liquid nitrogen. After thawing, the proteins were separated by SDS-PAGE and the protein bands were excised from the gel. Radioactivity bound to CheF:D729K was quantified by liquid scintillation counting. The CheF:D729K-Pi to CheY ration was 1:1 (…), 5:1 (z), 10:1 (|) and 20:1 (‹). Each

data point is the mean of two independent measurements.

Similar curves were obtained when CheV2 was present in the reaction mixture instead of CheY (Fig. 35).

Fig. 35. Loss of label from CheF:D729K-Pi (2.8 µM) to CheV2. Reaction conditions as

in Fig. 34. The CheF:D729K-Pi to CheV2 ration was 1:1 (), 5:1 (…) and 10:1 (|).

Red curve: radiolabel bound to kinase in absence of response regulator (under assay conditions). Each data point is the mean of two independent measurements. Data were normalized to data from Fig. 34 as basis.

Again, label was transferred to the response regulators, whereas in their absence, the label remained bound to the kinase (red curve in Fig. 35). Under the assumption that the dephosphorylation of the response regulator phosphates become rate-limiting for the dephosphorylation reaction of CheF:D729K-Pi, the CheY and CheV2 concentrations were

successively lowered to ensure pseudo-first order conditions for the efflux of label from CheF:D729K-Pi. In the case of the reaction catalyzed by

CheY, the life-time of CheY-phosphate can be determined from the linear parts of the curves (Fig. 34; 10 and 20 fold excess of CheF; 30 to 120 sec of experiment). This life-time is 14 ± 1 sec. As expected, the reaction proceeds considerably slower with CheV2 than with CheY. Even at equimolar concentrations of both proteins, the transfer of phosphate groups from the kinase to CheV2 takes more than 180 sec, and therefore the life-time of CheV2 phosphate can not be calculated from these data. Residual radioactivity remains bound to the kinase in both cases that is not transferred to the response regulators. Interestingly, between t≈20 and 30 sec, the curves apparently indicate a reverse flow of phosphate groups from the response regulator phosphates to the kinase.