Human L­asparaginase: Acquiring knowledge of its activation (Review).

L­asparaginase enzymes have been a vital component of acute lymphoblastic leukemia therapy for >40 years. L­asparaginase acts by depleting plasma L­asparagine, which is essential to the survival of leukemia cells. In contrast to normal cells, tumor cells cannot synthesize L­asparagine and thus depend on its external uptake for growth. Currently, three bacterial L­asparaginases are used in therapy; however, they are associated with severe side­effects related to high toxicity and immunogenicity. The introduction of human L­asparaginase­like protein 1 in acute lymphoblastic leukemia treatment would avoid the problems caused by the bacterial enzymes; however, a major difficulty in the therapeutic use of the human enzyme comes from the fact that human L­asparaginase must be activated through an autoprocessing step, which is a low­efficiency process in vitro that results in reduced enzymatic activity. The present review article aimed to contribute to the understanding of the enzyme self­activation process and focuses on the efforts made for the development of a therapeutic variant of human L­asparaginase.

In-solution behavior and protective potential of asparagine synthetase A from Trypanosoma cruzi.

l-Asparagine synthetase (AS) acts in asparagine formation and can be classified into two families: AS-A or AS-B. AS-A is mainly found in prokaryotes and can synthetize asparagine from ammonia. Distinct from other eukaryotes, Trypanosoma cruzi produces an AS-A. AS-A from Trypanosoma cruzi (Tc-AS-A) differs from prokaryotic AS-A due to its ability to catalyze asparagine synthesis using both glutamine and ammonia as nitrogen sources. Regarding these peculiarities, this work uses several biophysical techniques to provide data concerning the Tc-AS-A in-solution behavior. Tc-AS-A was produced as a recombinant and purified by three chromatography steps. Circular dichroism, dynamic light scattering, and analytical size exclusion chromatography showed that Tc-AS-A has the same fold and quaternary arrangement of prokaryotic AS-A. Despite the tendency of protein to aggregate, stable dimers were obtained when solubilization occurred at pH ≤ 7.0. We also demonstrate the protective efficacy against T. cruzi infection in mice immunized with Tc-AS-A. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model.

L-Asparaginase from Erwinia carotovora: insights about its stability and activity.

Enzymatic prospection indicated that L-asparaginase from Erwinia carotovora (ECAR-LANS) posses low glutaminase activity and much effort has been made to produce therapeutic ECAR-LANS. However, its low stability precludes its use in therapy. Herein, biochemical and biophysical assays provided data highlighting the influence of solubilization and storage into ECAR-LANS structure, stability, and activity. Moreover, innovations in recombinant expression and purification guaranteed the purification of functional tetramers. According to solubilization condition, the L-asparaginase activity and temperature of melting ranged up to 25-32%, respectively. CD spectra indicate the tendency of ECAR-LANS to instability and the influence of ß-structures in activity. These results provide relevant information to guide formulations with prolonged action in the bloodstream.