Mutations in asparaginase II from E. coli and implications for inactivation and PEGylation

Biophys Chem. 2023 Aug:299:107041. doi: 10.1016/j.bpc.2023.107041. Epub 2023 May 13.


All clinically-used asparaginases convert L-asparagine (L-Asn) to l-aspartate (L-Asp) and l-glutamine (L-Gln) to L-glutamate (L-Glu), which has been useful in reducing bioavailable asparagine and glutamine in patients under treatment for acute lymphoblastic leukemia. The E. coli type 2 L-asparaginase (EcA2) can present different sequences among varying bacterial strains, which we hypothesized that might affect their biological function, stability and interchangeability. Here we report the analysis of two EcA2 provided by the public health system of a middle-income country. These enzymes were reported to have similar specific activity in vitro, whereas they differ in vivo. Protein sequencing by LC-MS-MS and peptide mapping by MALDI-ToF-MS of their tryptic digests revealed that Aginasa™ share similar sequence to EcA2 from E. coli strain BL21(DE3), while Leuginase™ has sequence equivalent to EcA2 from E. coli strain AS1.357. The two amino acid differences between Aginasa™ (64D and 252 T) and Leuginase™ (64 N and 252S) resulted in structural divergences in solution as accessed by small-angle X-ray scattering and molecular dynamics simulation trajectories. The conformational variability further results in dissimilar surface accessibility with major consequences for PEGylation, as well as different susceptibility to degradation by limited proteolysis. The present results reveal that the sequence variations between these two EcA2 variants results in conformational changes associated with differential conformational plasticity, potentially affecting physico-chemical and biological properties, including proteolytic and immunogenic silent inactivation.

Keywords: Enzyme mechanism; L-Asparaginase 2; Leukemia; PEGylation; Protein dynamics; Stability; cancer therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Asparaginase* / chemistry
  • Escherichia coli / genetics
  • Humans
  • Mutation
  • Polyethylene Glycols* / chemistry
  • Polyethylene Glycols* / metabolism
  • Precursor Cell Lymphoblastic Leukemia-Lymphoma*


  • Asparaginase
  • Polyethylene Glycols