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Generalized enzymatic mechanism of catalysis by tetrameric L-asparaginases from mesophilic bacteria

  1. Author:
    Strzelczyk,Pawel
    Zhang,Di
    Dyba,Marzena
    Wlodawer,Alexander
    Lubkowski,Jacek

  2. Author Address

    NCI, Macromol Crystallog Lab, Frederick, MD 21701 USA.NCI, Basic Sci Program, Struct Biophys Lab, Frederick Natl Lab Canc Res, Frederick, MD 21701 USA.
    1. Year: 2020
    2. Date: OCT 15
  2. Journal: SCIENTIFIC REPORTS
  3. NATURE RESEARCH,
    1. 10
    2. 1
  5. Type of Article: Article
  6. Article Number: 17516
  7. ISSN: 2045-2322
  1. Abstract:

    The mechanism of catalysis by the l-glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of l-Asn by the type II l-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme. Based on results presented in this report, we postulate that all homotetrameric l-asparaginases from mesophilic bacteria utilize a common ping-pong mechanism of catalysis consisting of two subsequent nucleophilic substitutions. Several new structures of non-covalent complexes of PGA with different substrates, as well as structures of covalent acyl-enzyme intermediates of PGA with canonical substrates (l-Asp and l-Glu) and an opportunistic ligand, a citrate anion, were determined. The results of kinetic experiments monitored by high-resolution LC/MS, when combined with new structural data, clearly show that the reaction catalyzed by l-glutaminase-asparaginases proceeds through formation of a covalent intermediate, as observed previously for EcAII. Additionally, by showing that the same mechanism applies to l-Asn and l-Gln, we postulate that it is common for all these structurally related enzymes.

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External Sources

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  2. DOI: 10.1038/s41598-020-74480-4
  3. View record in Web of Science®
  4. WOS: 000582705900130

Library Notes

  1. Fiscal Year: FY2020-2021
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