Rescue of catalytically impaired enzyme mutants is well established, but not as observed for PaATPPRT. Chemical rescue by small molecules that mimic missing residue side chains is a useful tool to probe the function of active-site residues in catalysis, and a hyper-nucleophilic cholesterol analogue in which an -OOH group replaces the sterol -OH group could rescue the base-catalysed endoproteolytic activity of a mutant hedgehog protein where the catalytically essential aspartate general base was mutated to an alanine, which rendered the reaction highly impaired with the natural substrate. A catalytically compromised receptor tyrosine kinase carrying mutations in the activation loop tyrosine residues that would otherwise be autophosphorylated could be allosterically rescued by the juxtamembrane segment via autophosphorylation of this segment's Y687, but in this case the effect of the phospho-Y687 is exerted by direct interaction with arginine residues that normally interact with the phosphorylated tyrosine residues in the activation loop. In heterotetrameric tryptophan synthase, where catalysis by the β subunit is allosterically activated by the  subunit, the catalytically inactive E109A mutant of the β subunit could not be rescued by the  subunit, but the activity of the E109A-2β2 complex could be partially restored by CsCl, possibly by modulation of the conformational ensemble of the complex. In human prolyl isomerase CypA, the second-shell S99T mutation, which is highly detrimental to catalysis, can be partially counteracted by additional mutations outside the active site which rescue the dynamics of interconversion between two essential conformations. PaATPPRT is unique because the rescue of catalytically compromised PaHisGS mutants by PaHisZ, and even by the orthologous AbHisZ, is truly allosteric since the regulatory subunit binds far from the active site where the mutations exert a detrimental effect on transition state stabilisation. The narrowing of the distribution of states sampled by the PaHisGS upon PaHisZ binding has a direct impact on the positioning and orientation of R32 and R56, which are better poised to facilitate leaving group departure by electrostatic stabilisation of the PPi negative charges. This implies allosteric activation of catalysis in PaATPPRT involves modulation of the conformational flexibility of the holoenzyme and electrostatic preorganisation of the active site. The catalytic recruitment of R32 and R56 in concert with Mg 2+ to stabilise PPi is reminiscent of that of arginine residues in adenylate kinase to promote phosphate transfer from ADP to AMP, where thermally equilibrated protein motions were also proposed to help achieve optimal electrostatic preorganisation. Interestingly, replacement of a key arginine residue for a lysine was detrimental to catalysis in adenylate kinase as well. Another important aspect of the allosterically rescued R32A-and R56A-PaATPPRT is that kcat is at least partially limited by the chemical step, a drastic change from WT-PaATPPRT in which kcat is determined by product release. Electrostatic preorganisation exerts its effect on catalysis at the chemical step, i.e. as the reaction progresses from the preorganised Michaelis complex to the transition state. Thus it is paramount that an experimentally measured rate constant purporting to reflect any coupling of protein motions to the preorganisation of the Michaelis complex be limited by the chemical step. This is what is observed with the allosteric rescue of the catalytically compromised mutants of PaHisGS, establishing a direct connection between PaHisZ-modulated rotamers of R32 and R56 and electrostatic preorganisation of the active site, which is required for optimal catalysis. Crystallisation, X-ray data collection and data processing. Crystals of R56A-PaHisGS were grown, soaked in PRPP and ATP and stored as described for WT-PaHisGS, whereas crystals of R56A-PaATPPRT were grown as described for WT-PaATPPRT and soaked in PRPP and ATP and stored as described for WT-PaATPPRT. X-ray diffraction data for R56A-PaHisGS were collected in house as previously reported and processed with iMosflm, Structures were refined using cycles of model building with COOT and refinement with Refmac. ATP was modelled at either 70% or 80% occupancy in R56A-PaATPPRT. Partial charges for the ligand PRPP were calculated in vacuo at the HF/6-31G* level of theory using Gaussian 16 Rev. A.03, and fitted using the standard restrained electrostatic potential (RESP) protocol as implemented in Antechamber (Supplementary Table ). All other force field terms for PRPP were then described using the Amber force field ff14SB together with revised parameters to describe bioorganic phosphates. The parameters for ATP were taken from the literature. We used an octahedral cationic dummy model to describe Mg 2+ , following from previous successful results using this model. All MD simulations were performed using the GPU-accelerated version of Amber16, with the protein and water molecules described using the amber force field ff14SB and the TIP3P water model, respectively. All systems were solvated in an octahedral box of water molecules, extended 8 Å from the closest solute molecule in every direction. Each system was neutralized by adding Na + or Cl -counterions to ensure overall charge neutrality. Counterions were placed using the "addions" approach as implemented in Amber16. The dimeric and hetero-octameric forms of the enzyme were simulated for 10  500 ns and 5  500 ns, respectively, in the NPT ensemble. The solvated systems were first minimized using 5,000 steps of steepest descent minimization with 500 kcal mol -1 Å -2 positional restraints placed on all solute atoms to minimize all hydrogen atoms and solvent molecules, followed by 5,000 steps of conjugate gradient minimization, with the restraint dropped to 5 kcal mol -1 Å -2 . The minimized system was then heated from 0 to 300 K in an NVT ensemble over 250 ps of simulation time using the Berendsen thermostat