## 2 Results and Discussion  
### Toxin \(\zeta\)Hydrolyses ATP in the Presence of UNAG  
Recently, it was documented that in the presence of ATP-Mg\({}^{2+}\), \(\zeta\) toxin, in the presence of \(\varepsilon_{2}\) antitoxin, phosphorylates the cell wall precursor UNAG by attaching a phosphoryl group to the 3'-hydroxyl group of the _N_-acetylglucosamine moiety to form UDP-_N_-acetylglucosamine-3'-phosphate (UNAG-3P) [9]. The low efficiency of this reaction, which was not stoichiometric, could be attributed to the presence of the antitoxin or because the phosphorylation reaction might be uncoupled from the ATPase reaction. In this work we have we tested whether \(\zeta\) toxin purified by a novel protocol, free of antitoxin (as described in the Experimental section), is able to hydrolyze ATP in the presence or absence of UNAG. We have also tested whether the hydrolysis reaction is coupled with the phosphotransfer reaction and if GTP could work as a donor of inorganic phosphate (P\({}_{i}\)) for the transfer reaction. The products were separated from the substrate by thin-layer chromatography (TLC) and analyzed by autoradiography. In the presence of UNAG, \(\zeta\) toxin hydrolyzed [\(\gamma^{32}\)P]-ATP, and we observed the accumulation of a radioactive product(s) which co-migrate with the front (data not shown). This was the expected position for the product of ATP hydrolysis, which is inorganic phosphate [\({}^{32}\)Pi]. We do not know how [\(\gamma^{32}\)P]-UNAG-3P should migrate under the conditions used, but one hypothesis is that it co-migrates with the TLC front under the experimental conditions used. To address this, we perform experiments with other TLCuents in order to separate [\({}^{32}\)Pi] from [\({}^{32}\)P]-UNAG-P, but we failed to have a significant separation of both of them under the conditions used (data not shown). Alternatively,the reaction is uncoupled and [\(\gamma^{32}\)P]-UNAG-P accounts only to a minor fraction of the radiolabelled product. To quantify the rate of ATP hydrolysis the experiments were performed using [\(\alpha^{32}\)P]-ATP. If \(\zeta\) hydrolyses [\(\alpha^{32}\)P]-ATP it should be converted onto [\(\alpha^{32}\)P]-ADP that runs faster than [\(\alpha^{32}\)P]-ATP in a TLC. The commercial radioactive [\(\alpha^{32}\)P]-ATP contains 6%-10% of [\(\alpha^{32}\)P]-ADP [17], hence this is our background level, and this was used in the TLC as an internal control that marked the respective running positions of substrates and products. In the absence of \(\zeta\) toxin no [\(\alpha^{32}\)P]-ATP hydrolysis was observed (Figure 1, lane 10). Purified \(\zeta\) toxin (0.5 \(\upmu\)M) did not hydrolyze [\(\alpha^{32}\)P]-ATP (0.5 mM) in the absence of UNAG, but \(\zeta\) toxin hydrolyzed \(>\)85% of the ATP substrate upon addition of UNAG (2 mM), in a 60 min reaction (Figure 1, lanes 1 and 2). When saturating ATP concentrations were used, also a large fraction of the ATP was hydrolyzed (Figure 1, lanes 3 and 4). At ATP:UNAG ratios of 2.5:1 or 5:1 \(\sim\)70% and \(\sim\)40% of radiolabelled [\(\alpha^{32}\)P]-ATP was converted onto [\(\alpha^{32}\)P]-ADP, respectively, in a 60 min reaction (Figure 1, lanes 3 and 4). Since the conversion of ATP onto ADP was significantly higher than if each P\({}_{i}\) generated was transferred to the 3'-OH group of the sugar moiety of UNAG, we tentatively assume that the reaction is not tightly coupled. Previously, it was shown that \(\zeta\) toxin, in the presence of ATP-Mg\({}^{2+}\) and the \(\varepsilon_{2}\) antitoxin, attaches a phosphoryl group to the 3'-hydroxyl group of the \(N\)-acetylglucosamine moiety to form UDP-\(N\)-acetylglucosamineamine-3'-phosphate (UNAG-3P) [9]. Hence we assume that \(\zeta\) toxin alone  
Figure 1: Uridine diphosphate-\(N\)-acetylglucosamine (UNAG)-dependent \(\zeta\) hydrolysis of ATP is poorly competed by GTP.