Biomedical Chemistry: Research and Methods 2019, 2(1), e00071

Physical-Chemical Properties of L-asparaginase Mutants from Rhodospirillum Rubrum which Showed Antitelomerase Activity

M.V. Pokrovskaya 1*, S.S. Aleksandrova1, A.V. Veselovsky1, D.D. Zdanov1,2, V.S. Pokrovsky1,2,
M.A. Eldarov3, D.V. Grishin1, Yu. A. Gladilina1, I.Yu. Toropigin1, N.N. Sokolov1

1Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia; *e-mail:
2Рeoples Friendship University of Russia, 6 Miklukho-Maklaya str., Moscow, 117198 Russia 3Research Center of Biotechnology RAS, 33 Leninsky Prospect, Moscow, 119071 Russia

Keywords: L-asparaginase Rhodospirillum rubrum; site-directed mutagenesis; physical-chemical properties


The whole version of this paper is available in Russian.

Rru_A3730 protein is a bacterial Rhodospirillum rubrum L-asparaginase (RrA), which is known by its anticancer activity. RrA variants with point amino acid substitutions in the region of 150 amino acids residues: RrAN17,K149E, RrAE149R,V150P,F151T, RrАN17,E149R,V150P, RrAE149R,V150P, showed antiproliferative properties, and also by their ability to suppress telomerase activity. This work is devoted to comparison of physical-chemical and catalytic properties of these mutant forms of RrA. It is shown that pH optimum is in the alkaline zone (8.5 – 9.3); L-glutaminase and D-asparaginase activity is respectively not more than 0.1% and 1.6% of L-asparaginase for all studied variants of RrA. The presence of the N17-terminal amino acid sequence MASMTGGQMGRGSSRQ of the capsid protein of bacteriophage T7 in the RrA structure leads to an increase in the thermal stability of mutant RrA analogues (from 50°C to 56°C) and their resistance to denaturation in the presence of 3 – 4 M urea. It is of Metal ions exhibit multidirectional effects on L-asparaginase activity of RrA. K+, Ca2+, Zn2+, Cs+, Co2+ in significantly affect the activity of L-asparaginase, while Mn2+, Cu2+, Fe3+ ions inhibit it. There was no correlation between antitelomerase (antiproliferative) activity and kinetic properties of mutant forms of L-asparaginase RrA.

Figure 1. Electrophoresis of the purified preparations of RrA mutant forms in 12% SDS-PAAG.
M – Molecular weights marker #SM0431
1 – RrA17N,K149E 9 μg
2 – RrА17N,E149R,V150P 20 μg
3 – RrАE149R,V150P 16 μg
4 – RrAE149R, V150P,F151T 18 μg
Figure 2. Dependence activity of L-asparaginase RrA variants on the ionic strength in 0.1 M Na-phosphate buffer pH 7.4 (A) and in 0.01 M NaHCO3 buffer pH 7.4 (B).
Figure 3. Dependence activity of L-asparaginase RrA variants on pH.
Figure 4. Influence of temperature on the L-asparaginase activity of RrA variants.
Figure 5. Thermal stability of RrA variants L-asparaginase.
Figure 6. Denaturation of RrA variants in urea solution, pH 7.4 (A) and pH 9.2 (B).

Table 1. Influence of metal ions and some reagents on the L-asparaginase activity of RrA mutant forms.


The authors express their deep gratitude to PhD Natalia F. Belyaeva (research Institute of biomedical chemistry
V. N. Orekhovich, Russia) for participation in discussion of results and Vasily N. Lazarev (research Institute of physical and chemical medicine, Russia) for DNA sequencing.


The work was performed in the framework of the Program for Basic Research of the State Academies of Sciences for 2013–2020 and partially supported by Russian Foundation for Basic Research grant 15-34-70020.


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