# Toxin \(\zeta\) Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool
## 2 Results
### Toxin \(\zeta\) Has a Low Affinity for UNAG  
To verify the affinity binding predictions, we examined the rate-limiting step(s) within the ATP hydrolysis cycle and performed classic Michaelis-Menten analysis to define the K\({}_{\text{m}}\), K\({}_{\text{cat}}\) and V\({}_{\text{max}}\) (Figure 4A-D). In _B. subtilis_, the intracellular ATP and UNAG pools approached \(\sim\)10 mM each in glucose-fed exponentially grown cells [13; 45; 46]. Similar concentrations were observed in exponentially _E. coli_ K-12 cells grown in LB medium [47]. The UNAG pool, however, is significantly lower (\(\sim\)1 mM) in _E. coli_ B cells grown in LB medium [48]. In the presence of variable ATP as the main substrate (0.03 to 10 mM) and physiological UNAG concentrations (10 mM), limiting toxin \(\zeta\) approached the maximal rate of ATP hydrolysis K\({}_{\text{cat}}\) of 1200 \(\pm\) 90 min\({}^{-1}\) (Figure 4A), with a K\({}_{\text{m}}\) for ATP of \(\sim\)0.6 mM (Figure 4C). This is consistent with the observation that toxin \(\zeta\) expression, at or near physiological concentration (\(\sim\)1 mM), reduces the ATP pool \(\sim\)2.8-fold.  
\begin{table}
\begin{tabular}{l c c c c c c c c} \hline
**Condition** & **wt** & **K46A** & **D67A** & **E100A** & **E116A** & **T128A** & **R158A** & **R171A** \\ \hline H-bonds & 9 & 1 & 2 & 4 & 3 & 3 & 2 & 4 \\ Contacts & 13 & 10 & 11 & 13 & 9 & 8 & 8 & 12 \\ Atom & 39 & 41 & 50 & 46 & 39 & 32 & 46 & 53 \\ Xscore & \(-\)7.36 & \(-\)6.77 & \(-\)6.87 & \(-\)7.09 & \(-\)8.81 & \(-\)7.00 & \(-\)7.17 & \(-\)7.23 \\ DSX & \(-\)99.592 & \(-\)74.601 & \(-\)85.754 & \(-\)77.066 & \(-\)79.611 & \(-\)85.631 & \(-\)83.521 & \(-\)84.271 \\ \hline \multicolumn{8}{l}{H-bonds, total number of H-bonds between UNAG and \(\zeta\) Contacts: number of amino acids making contacts with UNAG. Atom contacts, total number of contact atoms; Xscore, estimated binding energy in Kcal/mol (lower is better); DSX, DrugScore. In the two latter conditions the lower scores indicate stronger binding affinity.} \\ \end{tabular}
\end{table}
Table 2: Predicted interactions between UNAG and various \(\zeta\) mutants.  
In the presence of variable concentrations of UNAG (0.12 to 16 mM) and physiological ATP concentrations (10 mM), the rate of \(\zeta\)-mediated ATP hydrolysis approached the maximal rate of ATP hydrolysis with a K\({}_{\rm cat}\) of 1100 \(\pm\) 75 min\({}^{-1}\) (Figure 4B), with a K\({}_{\rm m}\) for UNAG of \(\sim\)3 mM (Figure 4D). Toxin \(\zeta\) had a \(\sim\)6-fold higher catalytic efficiency at 8 mM ATP (3.4 \(\times\) 10\({}^{4}\) M\({}^{-1}\) s\({}^{-1}\)) than at 16 mM UNAG (5.7 \(\times\) 10\({}^{3}\) M\({}^{-1}\) s\({}^{-1}\)) (Figure 4C,D), being both, ATP and UNAG concentrations, above the K\({}_{\rm m}\) value. It is likely that the significant drop in the ATP pool upon toxin \(\zeta\) expression, its lower affinity for UNAG and the higher affinity of toxin \(\zeta\) for UNAG-P may compromise the transfer of the P\(\gamma\) of ATP to the O3' of UNAG and lead to a significant drop in the ATP pool. This is consistent with the affinity binding predictions that revealed relatively higher affinities for UNAG than for ATP (Table 1) and the decrease in vivo pool of the ATP pool upon toxin \(\zeta\) expression [13]. Some of these features are also observed in a distantly related \(\zeta\)-like toxin prevalent in Neisseria Gonorrhoeae plasmids (termed ng_\(\zeta\)1) [49]. This new subclass of \(\zeta\)-like toxin produces ADP in excess with respect to UNAG-P, and shows a catalytic efficiency \(\sim\)8-fold higher for ATP than for UNAG [49]. Toxin ng_\(\zeta\)1, however, phosphorylates the C4\({}^{\prime}\)-OH group of the N-acetylglucosamine moiety of UNAG rather than the C3\({}^{\prime}\)-OH group as shown for the Firmicutes toxins [18; 49].