Upon addition of excess PaHisZ, some PRATP formation could be marginally detected above the assay background noise (Fig. ), demonstrating R32A/R56A/K57A-PaATPPRT still retains residual catalytic activity. However, the apparent rate constant is reduced ~777fold in comparison with that of WT-PaATPPRT (Supplementary Fig. ), and ~340-fold and ~222-fold in comparison with those of R32A-PaATPPRT and R56A-PaATPPRT, respectively (Supplementary Fig. ), in accordance with the proposed necessity for at least one of the two arginine residues to aid in leaving group departure for full catalytic power of PaATPPRT.