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Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase
[Image: see text] Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisG(S)) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg(2+)-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical Society
2018
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128619/ https://www.ncbi.nlm.nih.gov/pubmed/29940105 http://dx.doi.org/10.1021/acs.biochem.8b00559 |
Sumario: | [Image: see text] Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisG(S)) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg(2+)-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N(1)-(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 (31)P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg(2+) by Mn(2+) 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(N)2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn(2+) 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. |
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