Electrospray ionisation/time-of-flight-mass spectrometry (ESI/TOF-MS) confirmed the molecular masses of WT-, C115A-, C115S-, D179A-, R32A-, R56A-, and R56A/K57A-PaHisGS variants were in agreement with the predicted values (Supplementary Fig. ). The introduction of the D179N mutation was confirmed by MS/MS analysis of tryptic fragments (Supplementary Fig. ). In an initial activity screen, reactions were monitored for just under 1 min by the continuous and direct UV/VIS absorbance-based assay for ATPPRT activity at fixed PRPP and ATP concentrations sufficient to saturate WT-PaHisGS. PRATP formation was readily detected with C115A-, D179A-, D179N-, and WT-PaHisGS (Fig. ), and linear regression of the data yielded apparent rate constants shown in Supplementary Table .