Skip to content

Research at St Andrews

Allosteric activation shifts the rate-limiting step in a short-form ATP phosphoribosyltransferase

Research output: Contribution to journalArticlepeer-review

Author(s)

Gemma Fisher, Catherine M. Thomson, Rozanne Stroek, Clarissa M Czekster, Jennifer S. Hirschi, Rafael G da Silva

School/Research organisations

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.
Close

Details

Original languageEnglish
Pages (from-to)4357-4367
JournalBiochemistry
Volume57
Issue number29
Early online date25 Jun 2018
DOIs
Publication statusPublished - 24 Jul 2018

    Research areas

  • ATP phosphoribosyltransferase, Allostery, Density-functional theory, Pre-steady-state kinetics, Viscosity effects, Kinetic mechanism

Discover related content
Find related publications, people, projects and more using interactive charts.

View graph of relations

Related by author

  1. Bypassing the requirement for aminoacyl-tRNA by a cyclodipeptide synthase enzyme

    Harding, C. J., Sutherland, E., Hanna, J., Houston, D. & Melo Czekster, C., 15 Jan 2021, In: RSC Chemical Biology. Advance article, 11 p.

    Research output: Contribution to journalArticlepeer-review

  2. Dissecting the mechanism of (R)-3-hydroxybutyrate dehydrogenase by kinetic isotope effects, protein crystallography, and computational chemistry

    G. Machado, T. F., Purg, M., McMahon, S., Read, B., Oehler, V., Åqvist, J., Gloster, T. & da Silva, R. G., 18 Dec 2020, In: ACS Catalysis. 10, 24, p. 15019–15032

    Research output: Contribution to journalArticlepeer-review

  3. Robust estimation of bacterial cell count from optical density

    iGEM Interlab Study Contributors, Melo Czekster, C. & Powis, S. J., 17 Sep 2020, In: Communications Biology. 3, 29 p., 512.

    Research output: Contribution to journalArticlepeer-review

  4. The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling

    Athukoralage, J. S., Graham, S., Rouillon, C., Gruschow, S., M Czekster, C. & White, M., 27 Apr 2020, In: eLife. 9, 16 p., e55852.

    Research output: Contribution to journalArticlepeer-review

  5. An exceptionally potent inhibitor of human CD73

    Bowman, C. E., da Silva, R. G., Pham, A. & Young, S. W., 6 Aug 2019, In: Biochemistry. 58, 31, p. 3331-3334 4 p.

    Research output: Contribution to journalArticlepeer-review

Related by journal

  1. An exceptionally potent inhibitor of human CD73

    Bowman, C. E., da Silva, R. G., Pham, A. & Young, S. W., 6 Aug 2019, In: Biochemistry. 58, 31, p. 3331-3334 4 p.

    Research output: Contribution to journalArticlepeer-review

  2. Insights into the mechanism of the cyanobactin heterocyclase enzyme

    Ge, Y., Czekster, C. M., Miller, O. K., Botting, C. H., Schwarz-Linek, U. & Naismith, J. H., 23 Apr 2019, In: Biochemistry. 58, 16, p. 2125-2132 8 p.

    Research output: Contribution to journalArticlepeer-review

  3. Mapping the structural path for allosteric inhibition of a short-form ATP phosphoribosyltransferase by histidine

    Thomson, C. M., Alphey, M. S., Fisher, G. & da Silva, R. G., 16 Jul 2019, In: Biochemistry. 58, 28, p. 3078-3086

    Research output: Contribution to journalArticlepeer-review

  4. Linear Eyring plots conceal a change in rate-limiting step in an enzyme reaction

    Machado, T. F. G., Gloster, T. & da Silva, R. G., 11 Dec 2018, In: Biochemistry. 57, 49, p. 6757-6761

    Research output: Contribution to journalArticlepeer-review

ID: 255057069

Top