This mimics the natural condition in a crowded cellular environment, where the protein is exposed to many different random forces. Thus, the DFI analysis aims to capture the contribution of each position in the protein to the underlying functional dynamics, highlighting the key residues and regions that mediate functionally important dynamical information. Our analysis (Supplementary Fig. ) reveal PaHisZ binding changes the DFI of PaHisGS compared with nonactivated PaHisGS, in particular at the helices inferred to be important for communication at the PaHisGS-PaHisZ interface from the SPM analysis (Fig. ), as well as the region containing R32 and R56. Given the catalytic importance of R32 and R56 and their hypothesized involvement in facilitating leaving group departure, we tracked the distance between Cζ of each side chain and P of the PPi moiety of PRPP during our simulations (Fig. ). It can be gleaned from the data that the R56 side chain displays a bimodal distribution of distances in WT-PaHisGS (Fig. ). This distribution comprises a peak at ~4.4 Å corresponding to a catalytic conformation in which this side chain forms a salt-bridge with the PRPP PPi moiety, and another at ~7.8 Å, corresponding to a non-catalytic rotamer of this residue. Binding of PaHisZ shifts the distance distribution towards the catalytically active rotamer (Fig. ). This furnishes support for PaHisZ binding constraining the conformational dynamics of PaHisGS, fostering a preorganized active site in which the R56 guanidinium group is poised to help stabilize the leaving group at the transition state. increases WT-PaHisGS kcat, and density-functional theory calculations provided a rationale for this effect based on more efficient stabilisation of the negative charges by Mn 2+ via dorbital bonding to the oxygens of the departing PPi at the transition state. This was corroborating evidence that chemistry was the rate-limiting step in PaHisGS catalysis, but not in PaATPPRT where Mn 2+ had no significant impact on kcat. This observation is reproduced here. At saturating concentrations of both substrates, Mn 2+ allows product formation to be detected at a WT-PaHisGS concentration too low to detect reaction with Mg 2+ , but does not increase the WT-PaATPPRT reaction rate (Fig. ). In contrast, when R32A-PaATPPRT and R56A-PaATPPRT reactions were carried out with Mn 2+ instead of Mg 2+ , the rate of product formation increased (Fig. ), and the apparent first-order rate constants increased by ~5-fold and ~3-fold, respectively, in comparison with those with Mg 2+ (Fig. ). This indicates the rates of the rescued mutants reflect the chemical step of the reaction, i. e. unlike WT-PaATPPRT, chemistry is at least partially rate-limiting for R32A-PaATPPRT and R56A-PaATPPRT. Interestingly, R32A-PaATPPRT and R56A-PaATPPRT apparent first-order rate constants with Mn 2+ are even higher than the corresponding one for WT-PaATPPRT.