Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg²⁺-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N¹-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisG_S is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisG_S, but steady-state kinetics and ³¹P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg²⁺ by Mn²⁺ enhances catalysis by HisG_S but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an S_N2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn²⁺ increasing HisG_S reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisG_S’s catalytic rate, but decreases the hetero-octamer’s, indicating that chemistry and product release are rate-limiting for HisG_S and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisG_S. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisG_S, accelerating chemistry beyond the product release rate.