Given the proposed role of R56 in stabilising the departure of the negatively charged leaving group, the possibility that a lysine residue could replace R56 with similar catalytic ability was considered. To evaluate this possibility, R56K-PaHisGS was produced. ESI/TOF-MS analysis resulted in the expected mass (Supplementary Fig. ), and DSF showed the mutation does not change the Tm of the protein (Supplementary Fig. ). At substrate concentrations saturating for WT-PaHisGS, however, the R56K-PaHisGS reaction rate is reduced ~24-fold in comparison with the WT-PaHisGS, and although there is an ~11-fold activation in the presence of PaHisZ, the R56K-PaATPPRT reaction rate is still ~2-fold lower compared with the nonactivated WT variant (Supplementary Fig. ). These observations indicate the amino group cannot substitute for the guanidinium group at position 56 of PaHisGS. Furthermore, allosteric activation by PaHisZ cannot rescue catalysis in this case.