Abstract
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 HisGS 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 HisGS, but steady-state kinetics and 31P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg2+ by Mn2+ enhances catalysis by HisGS but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an SN2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn2+ increasing HisGS reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisGS’s catalytic rate, but decreases the hetero-octamer’s, indicating that chemistry and product release are rate-limiting for HisGS 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 HisGS. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisGS, accelerating chemistry beyond the product release rate.
Highlights
Allosteric control of catalysis is a widespread strategy evolved in biosynthetic pathways.[1−4] The modulation of biochemical pathways for synthetic biology applications often requires overcoming or manipulating allosteric regulation.[5,6]
We recently reported several crystal structures of the psychrophilic bacterium Psychrobacter arcticus dimeric hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) (PaHisGS) and hetero-octameric adenosine 5′-triphosphate phosphoribosyltransferase (ATPPRT) holoenzyme (PaATPPRT),[26,29] from which an activation mechanism was inferred that involves tightening of the PaHisGS dimer in the hetero-octamer when both substrates are bound (Figure 1), which facilitates leaving group stabilization at the transition state.[29]
A steadystate ordered kinetic mechanism in which adenosine 5′-triphosphate (ATP) is the first substrate to bind to the enzyme, and PRATP is the last product to dissociate from it, has long been demonstrated for HisGL ATPPRTs.[21,22]
Summary
PaATPPRT initial rates were measured at saturating concentrations of one substrate and varying concentrations of the other, either ATP (0.4−5.6 mM) or PRPP (0.1−2.0 mM). PaATPPRT and PaHisGS initial rates were measured at saturating concentrations of one substrate and varying concentrations of the other, either ATP (0.4−5.6 mM) or PRPP (0.1−2.0 mM), in the presence of 0%, 18%, and 27%. Initial rates for PaATPPRT were measured in the presence of varying ATP (0.4−5.6 mM) and PRPP (0.1−2.0 mM), with 1 μM PaHisGS and 20 μM PaHisZ. Thermal denaturation assays (50 μL) for 7.5 μM PaHisGS were measured in the presence and absence of ligands (6 mM ATP, 2 mM PRPP, 208 μM PRATP, 3.6 mM PPi), with or without 22% glycerol (v/v) (apoenzyme) in 100 mM tricine, 100 mM. Three-dimensional plot of (E) PaATPPRT and (F) PaHisGS initial rate data, where lines are data fitting to eq 6
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