Abstract

For the experimental study of the role of the R65 residue in the stabilization of β-lactamases with the substitution of M182T, predicted by the analysis of the residue interaction networks (RINs), homogeneous preparations of the recombinant TEM-type β-lactamases with substitutions of R65L, M182A, and a combination of R65L and M182T mutations were obtained. The kinetic parameters of these enzymes were determined for penicillin, ceftazidime, cephalothin and CENTA. None of the investigated substitutions changed the substrate specificity of enzymes against β-lactam antibiotics. The substitution R65L leads to a decrease in thermal stability; the substitution of M182A and the combination of substitutions of R65L and M182T improve the thermal stability of β-lactamase in comparison with the wild-type enzyme TEM-1. Using differential scanning calorimetry, it was determined the enthalpy (ΔH, kJ/mol) and the denaturation temperature (T_mp, °C) for β-lactamases TEM-1, TEM(M182A), TEM-135(M182T), and TEM (R65L + M182T), which are, respectively, 554.0 and 50.8, 573.7 and 51.7, 654.4 and 55.9, and 647.4 and 51.9. The hypothesis of molecular mechanism, explaining the stabilizing role of M182T substitution in TEM type β‑lactamases, was supplemented by the effect of changing the conformation of the R65 residue and the appearance of its new contacts with the residues of the Ω-loop of β-lactamase.

中文翻译：

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残留R65对M182T取代的TEM型β-内酰胺酶稳定性的影响

摘要

通过对残基相互作用网络（RINs）的分析，预测了R65残基在M182T取代下稳定β-内酰胺酶中的作用的实验研究，均一制备了具有取代基的重组TEM型β-内酰胺酶获得了R65L，M182A的片段，以及R65L和M182T突变的组合。确定了这些酶的动力学参数，用于青霉素，头孢他啶，头孢菌素和CENTA。所研究的替代均未改变酶对β-内酰胺抗生素的底物特异性。取代R65L导致热稳定性降低；与野生型酶TEM-1相比，M182A的取代以及R65L和M182T的取代的组合提高了β-内酰胺酶的热稳定性。使用差示扫描量热法分别为β-内酰胺酶TEM-1，TEM（M182A），TEM-135（M182T）和TEM（R65L + M182T）的H（kJ / mol）和变性温度（T _mp，°C）， 554.0和50.8、573.7和51.7、654.4和55.9、647.4和51.9。分子机制假说解释了M182T取代在TEM型β-内酰胺酶中的稳定作用，并通过改变R65残基的构象以及其与β的Ω环残基新接触的出现而得到补充。 -内酰胺酶。