Feller G, Bussy O, Houssier C, Gerday C
Laboratory of Biochemistry, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium.
J Biol Chem. 1996 Sep 27;271(39):23836-41. doi: 10.1074/jbc.271.39.23836.
Chloride is the allosteric effector of vertebrate pancreatic and salivary alpha-amylases and of the bacterial alpha-amylase from Alteromonas haloplanctis. Activation experiments of A. haloplanctis alpha-amylase by several monovalent anions show that a negative charge, not restricted to that of Cl-, is essential for the amylolytic reaction. Engineering of the chloride binding site reveals that a basic residue is an essential component of the site. The mutation K337R alters the Cl--binding properties, whereas the mutation K337Q produces an active, chloride-independent enzyme. Comparison of the Kd values for Cl- in three homologous alpha-amylases also indicates that the binding affinity is dependent on the chloride coordination mode by this basic residue. Analysis of substrate and chloride binding according to the allosteric kinetic model shows that the chloride effector is not involved in substrate binding. By contrast, the pH dependence of activity and experiments of chemical modifications and Ca2+ inhibition show that the chloride ion is responsible for the pKa shift of catalytic groups and interacts with active site carboxyl groups.
氯离子是脊椎动物胰腺和唾液α-淀粉酶以及来自嗜盐交替单胞菌的细菌α-淀粉酶的别构效应物。几种单价阴离子对嗜盐交替单胞菌α-淀粉酶的激活实验表明，对于淀粉分解反应而言，并非仅限于氯离子的负电荷是必不可少的。氯离子结合位点的工程改造表明，一个碱性残基是该位点的重要组成部分。K337R突变改变了氯离子结合特性，而K337Q突变产生了一种活性的、不依赖氯离子的酶。三种同源α-淀粉酶中氯离子的解离常数（Kd）值比较也表明，结合亲和力取决于该碱性残基的氯离子配位模式。根据别构动力学模型对底物和氯离子结合的分析表明，氯离子效应物不参与底物结合。相比之下，活性的pH依赖性以及化学修饰和钙离子抑制实验表明，氯离子导致催化基团的pKa位移，并与活性位点羧基相互作用。
