Chen Zhijie, Fu Yu, Liu Shimeng, Huang Xinyu, Kong Xiaoting, Mao Zhaojie, Hu Ning, Zhang Fengxiang, Han Caijing
School of Pharmaceutical Sciences, Tsinghua University, 100084, Beijing, China.
School of Public Health, Weifang Medical University, Weifang, 261053, Shandong, China.
Heliyon. 2023 Jan 28;9(2):e13133. doi: 10.1016/j.heliyon.2023.e13133. eCollection 2023 Feb.
Aspartokinase (AK) is synergistically suppressed by Thr and Lys in the metabolic pathway. Site-directed mutations can significantly improve AK inhibition. Our previous studies confirmed that sites 379 and 380 were important sites affecting enzyme activity, so we further screen the double mutants with excellent enzymatic properties from sites 379 and 380, and discuss the difference of enzyme activity between the double mutants and single mutants. Here, a double mutant, T379L/A380 M, with improved enzyme activity (2.74-fold) was obtained. Enzymatic property experiments showed that the optimum temperature of T379L/A380 M increased from 26 °C (recombinant ; WT-AK) to 45 °C and that the optimal pH decreased from 8.0 (WT-AK) to 7.5. Further, the half-life decreased from 4.5 to 3.32 h. These enzymatic properties were better than other mutant strains. Inhibition was diminished with low concentrations of Lys, and Lys + Thr presented an activating role. Subsequently, the reasons for the improved AK enzyme activity were illustrated with microscale thermophoresis (MST) experiments and molecular dynamic (MD) simulation by measuring ligand affinity and AK conformational changes. MST showed that the affinity between T379L/A380 M and Lys decreased, but the affinity between T379L/A380 M and Asp increased, promoting T379L/A380 M enzyme activity. MD experiments showed that T379L/A380 M enhanced the Asp-ATP affinity and catalyzed the transfer of residues S192 and D193 to Asp, promoting T379L/A380 M enzyme activity. However, the mutation did not cause fluctuations in the substrate Asp and ATP pockets. This might be why the enzyme activity was inferior to that of the single mutants (T379L and A380 M).
天冬氨酸激酶（AK）在代谢途径中受到苏氨酸和赖氨酸的协同抑制。定点突变可显著改善对AK的抑制作用。我们之前的研究证实，379位和380位是影响酶活性的重要位点，因此我们进一步从379位和380位筛选出具有优异酶学性质的双突变体，并讨论双突变体与单突变体之间酶活性的差异。在此，获得了一种酶活性提高（2.74倍）的双突变体T379L/A380M。酶学性质实验表明，T379L/A380M的最适温度从26℃（重组野生型AK）提高到45℃，最适pH从8.0（野生型AK）降至7.5。此外，半衰期从4.5小时降至3.32小时。这些酶学性质优于其他突变菌株。低浓度赖氨酸可减弱抑制作用，赖氨酸+苏氨酸呈现激活作用。随后，通过微量热泳动（MST）实验和分子动力学（MD）模拟，通过测量配体亲和力和AK构象变化，阐明了AK酶活性提高的原因。MST表明，T379L/A380M与赖氨酸之间的亲和力降低，但T379L/A380M与天冬氨酸之间的亲和力增加，从而促进了T379L/A380M的酶活性。MD实验表明，T379L/A380M增强了天冬氨酸-ATP亲和力，并催化残基S192和D193向天冬氨酸的转移，从而促进了T379L/A380M的酶活性。然而，该突变并未导致底物天冬氨酸和ATP口袋的波动。这可能就是该酶活性低于单突变体（T379L和A380M）的原因。
