Martín J, Prieto I, Barbero J L, Pérez-Gil J, Mancheño J M, Arche R
Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, Madrid, Spain.
Biochim Biophys Acta. 1990 Feb 9;1037(2):133-9. doi: 10.1016/0167-4838(90)90158-c.
The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters Km and kcat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met----Ala168 mutation unstabilizes the first transition-state (E..S not equal to) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site.
通过寡核苷酸定点诱变将嗜柠檬酸克雷伯氏菌青霉素酰化酶中的第168位甲硫氨酸残基替换为丙氨酸。第168位丙氨酸突变体表现出与野生型不同的底物特异性，并增强了热稳定性。通过稳态动力学参数Km和kcat对温度的依赖性，获得了两种酶催化青霉素G水解的热力学曲线。为底物结合所确定的高活化焓和熵值表明发生了类似诱导契合的机制。第168位甲硫氨酸到丙氨酸的突变使第一个过渡态（E..S不等于）和酶-底物复合物（ES）不稳定，导致酶的缔合平衡和特异性常数降低。然而，在酰基酶形成过程中未观察到变化。得出的结论是，第168位残基参与了由底物酸性部分在活性位点的相互作用引起的酶构象重排。
