1-C.1). The addition of MgATP to the OppA mutants led to an increase in ATPase activity in a dose-dependent and saturable manner. The data of ATP hydrolysis were fed into Michaelis-Menten
equation. In nonlinear regression analysis the Michaelis constant, Km for the recombinant OppAR was 0.46 ± 0.04 mM ATP, whereas Km for the wild type selleck chemicals llc OppAWT was 0.18 ± 0.04 mM. As the Michaelis constant behaves reciprocally to the enzyme affinity this exhibits a higher affinity of OppAWT for ATP than OppAR. This may be due to a partial misfolding of the recombinant variant. However, the maximum reaction rate (Vmax 1543 ± 32.54 nmol/min/mg) was similar for both proteins. Figure 2 ATPase activity and adhesion of M. hominis membrane proteins P50, P60/P80 and OppA variants. ATPase activities of purified proteins (0.5 μg/well) were measured in the ammonium molybdate assay as a function of ATP concentration [A.1-C.1] Protein adhesion to HeLa cells was measured in cell-ELISA [A.2-C.2]. A comparison of the relative ATPase activity SC75741 manufacturer (black bars) and adhesion (striped bars) with regard to wild type OppA is shown in [A.3-C.3]. Data represent means of three independent experiments with triplicate samples in each experiment. Statistical analysis was performed by unpaired t-test and statistically significant results designated
by *. *P < 0.05, **P < 0.01, and ***P < 0.001. The ATPase activity or adhesion of the OppA mutants were compared with those of the recombinant OppA (R). As shown in Figure 2A.1 dephosphorylation of OppA had no influence on for its ATPase activity (Km 0.39 ± 0.04 mM ATP) whereas mutations within either the Walker A or Walker B motifs led to a dramatic decrease in ATP-hydrolysis. As previously shown in 2004  a single point mutation in the Walker A motif (K875R) led to a decreased ATP-hydrolysis by OppAWA1 to 15% whereas ATP-binding still occurred. Mutation of the whole Walker A motif in OppAWA2 resulted in the selleck chemical complete inhibition of both ATP-binding and hydrolysis. Exchanging the Walker A motif of M.
hominis with the putative Walker A sequence of M. pulmonis in OppAWA3 also led to inhibition of the ATP-hydrolysis indicating that the Walker A motif of M. pulmonis in this context is non-functional. As expected both the OppA-mutant lacking the Walker B motif (OppAΔWB) as well as the OppAN -mutant with a complete deletion of the C- terminal half of OppA, including the ATP-binding domain, did not show any ATPase activity (Figure 2C.1). Next we examined the contribution of the other conserved regions on the catalytic function of OppA. Deletion of the CS2 region (AA365-372) led to an increased Km in the OppAΔCS2mutant (2.56 ± 0.43 mM ATP) (Figure 2B.1). With regard to the OppAΔCs1 and OppAΔCs3 mutants the lowest affinity for ATP was observed for the OppAΔCs3 mutant (Km 2.86 ± 0.