Structural Features Underlying the Mismatch Between Catalytic and Cytostatic Properties in L-Asparaginase from Rhodospirillum rubrum

The underlying structural features of the mismatch between catalytic and cytostatic properties in L-asparaginase from Rhodospirillum rubrum (RrA) and three of its mutants were investigated. The rationale for selecting the specific mutations (RrAA64V, E67K; RrAR118H, G120R; RrAE149R, V150P, F151T) is to elucidate the role of inter-subunit interaction in RrA and its impact on catalytic efficiency and stability. Bioinformatic modeling revealed a predominantly negative surface charge on RrA with limited positive charge clusters in the vicinity of the interface region. Thus, some negatively charged groups were replaced with positively charged ones to enhance the electrostatic interactions and stabilize the enzyme quaternary structure. RrAA64V, E67K and RrAR118H, G120R additionally contained an N-terminal 17-amino acid capsid peptide derived from the bacteriophage T7 (MASMTGGQQMGRGSSRQ), which could potentially affect the conformational stability of theenzymes. Circular dichroism (CD) spectroscopy was applied to the kinetic parameters analysis of Asn hydrolysis and showed that native RrA displayed a Vmax of 30 U/mg and a KM of 4.5 ± 0.5 mM. RrAE149R, V150P, and F151T exhibited a substantially increased Vmax of 57 U/mg. The catalytic efficiency of Vmax/KM also improved compared to the native enzyme: the Vmax/KM increased from approximately 7 U/mg × mM−1 (for the native enzyme) to 9 U/mg × mM−1 for Mut3. Other mutants exhibited less pronounced changes. Thermo-denaturation studies allowed us to determine the phase transition parameters of the RrA variants in comparison with commercial reference sample EcA. RrAA64V, E67K and RrAR118H, G120R exhibited the most favorable phase transition parameters, with melting temperatures (Tm) of 60.3 °C and 59.4 °C, respectively, exceeding that of the wild-type RrA (54.6 °C) and RrAE149R, V150P, F151T (52 °C). The EcA demonstrated a slightly superior thermal stability, with a Tm of 62 °C. The mutations showed a significant effect on protein stability during trypsinolysis. Therefore, RrAE149R, V150P, F151T showed higher resistance (45% activity remaining after 30 min of trypsin exposure) compared to the native RrA retained 20% activity. EcA preparations exhibited lower stability to trypsinolysis (losing over 90% activity in 15 min). The cytostatic effects were evaluated using MTT assays against K562 (leukemic) and A549 (lung carcinoma) cell lines. The MTT assays with K562 cells revealed that RrAE149R, V150P, F151T (IC50 of 10 U/mL) and RrAR118H, G120R (IC50 of 11.5 U/mL) exhibited superior antiproliferative activity compared to native enzymes RrA (IC50 of 15 U/mL) and EcA (24 U/mL). RrAE149R, V150P, F151T showed the most significant improvement in cytostatic activity. The results obtained indicate that the substitutions in RrAE149R, V150P, F151T resulted in the improvement of the enzyme biocatalytic properties and an increase in the resistance to aggregation and trypsinolysis. This highlights the role of electrostatic interactions in stabilizing the oligomeric structure of the enzyme, which eventually translates into an improvement in cytostatic efficiency and antiproliferative forces.