Conversion of allosteric inhibition to activation in phosphofructokinase by protein engineering
Conversion of allosteric inhibition to activation in phosphofructokinase by protein engineering
复制标题
通过蛋白质工程将磷酸果糖激酶的变构抑制转化为激活
作者：
F. Lau;A. Fersht
许多酶受变构控制，通常具有结合到相同效应位点的抑制剂和激活剂。大肠杆菌中的磷酸果糖激酶就是这样一种酶，被磷酸烯醇丙酮酸（PEP）抑制，被ADP和GDP 1激活。个体与效应物的相互作用如何影响激活和抑制之间的平衡，特别是当两种配体共享相同结合位点时？我们发现，在效应位点的一个单一的残基突变，Glu→Ala 187，导致PEP是一个激活剂，而不是抑制剂。在低浓度的底物果糖-6-磷酸的情况下，突变体酶在毫摩尔浓度的PEP下比野生型酶活性高出一百倍以上。经典的Monod-Wyman-Changeux双态模型2过于简单，无法解释突变酶的特性。
Many enzymes are subject to allosteric control, often with inhibitors and activators binding to the same effector site. Phosphofructokinase in Escherichia coli is such an enzyme, being inhibited by phosphoenolpyruvate (PEP) and activated by ADP and GDP1. How do individual interactions with effectors affect the balance between activation and inhibition, especially when both ligands share aspects of the same binding site? We find that mutation of a single residue in the effector site, Glu→Ala 187, leads to PEP being an activator rather than an inhibitor. With low concentrations of the substrate fructose-6-phosphate, the mutant enzyme is more than one hundred times more active than wild-type enzyme at millimolar concentrations of PEP. The classical Monod–Wyman–Changeux two-state model2 is too simple to account for the properties of the mutant enzyme.