Maggi Maristella, Chiarelli Laurent R, Valentini Giovanna, Scotti Claudia
Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy; Department of Biology and Biotechnologies "Lazzaro Spallanzani", Laboratory of Protein Biochemistry, University of Pavia, Pavia, Italy.
Department of Biology and Biotechnologies "Lazzaro Spallanzani", Laboratory of Molecular Microbiology, University of Pavia, Pavia, Italy.
PLoS One. 2015 Feb 9;10(2):e0117025. doi: 10.1371/journal.pone.0117025. eCollection 2015.
Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.
四十多年来，细菌L-天冬酰胺酶一直被用作抗癌药物，尽管它在具有治疗效果的同时，由于其细菌来源以及似乎还因其二级谷氨酰胺酶活性而存在一些副作用。幽门螺杆菌II型L-天冬酰胺酶具有一些有趣的特性，其中与目前用于治疗的药物相比，它对L-GLN的催化效率较低。在本研究中，我们描述了一些酶变体，其催化活性和体外细胞毒性活性与野生型酶不同。特别是，催化苏氨酸（T16D和T95E）的替换使该酶的两种催化活性都丧失，再次强调了这些残基的重要作用。一个偶然的突变体M121C/T169M，对L-天冬酰胺的催化效率得以保留，但完全无法进行L-谷氨酰胺水解。有趣的是，该变体对HL-60细胞没有任何细胞毒性作用。M12C和T169M单突变体的催化活性降低（分别比野生型酶低近2.5至4倍）。突变体Q63E对天冬酰胺具有相似的催化效率，相对于野生型酶，其谷氨酰胺酶效率减半，当使用相似的天冬酰胺酶单位时，它能够发挥与野生型酶相当或更高的细胞毒性作用。这些发现可能与确定L-天冬酰胺酶的谷氨酰胺酶活性在药物抗增殖作用中的作用有关，并有助于阐明如何设计最佳的天冬酰胺酶/谷氨酰胺酶组合以实现不断改进的、针对患者的治疗。
