Hehir M J, Murphy J E, Kantrowitz E R
Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA.
J Mol Biol. 2000 Dec 8;304(4):645-56. doi: 10.1006/jmbi.2000.4230.
Escherichia coli alkaline phosphatase (EC 3.1.3.1) belongs to a rare group of enzymes that exhibit intragenic complementation. When certain mutant versions of alkaline phosphatase are combined, the resulting heterodimeric enzymes exhibit a higher level of activity than would be expected based upon the relative activities of the parental enzymes. Nine previously identified alkaline phosphatase complementation mutants were re-examined in this work in order to determine a molecular explanation of intragenic complementation in this experimental system. The locations of these mutations were determined by DNA sequence analysis after PCR amplification of the phosphatase-negative phoA gene. Most of the mutations involved ligands to metal-binding sites. Each of the mutant enzymes was re-created by site-specific mutagenesis, expressed, purified, and kinetically characterized. To investigate cooperativity between the two subunits, we analyzed heterodimeric forms of some of the site-specific mutant enzymes. To enable the isolation of the heterodimeric alkaline phosphatase in pure form, the overall charge of one subunit was altered by replacing the C-terminal Lys residue with three Asp residues. This modification had no effect on the kinetic properties of the enzyme. Heterodimeric alkaline phosphatases were created using two methods: (1) in vitro formation by dissociation at acid pH followed by reassociation at slightly alkaline pH conditions in the presence of zinc and magnesium ions; and (2) in vivo expression from a plasmid carrying two different phoA genes. Increases in k(cat), as well as a large reduction in the p-nitrophenyl phosphate K(m) were observed for certain combinations of mutant enzymes. These results suggest that the structural assembly of E. coli alkaline phosphatase into the dimer induces cooperative interactions between the monomers necessary for the formation of the functional form of the holoenzyme.
大肠杆菌碱性磷酸酶（EC 3.1.3.1）属于表现出基因内互补的稀有酶类。当某些碱性磷酸酶的突变体版本组合在一起时，产生的异二聚体酶表现出比基于亲本酶的相对活性预期更高的活性水平。在这项工作中，对九个先前鉴定的碱性磷酸酶互补突变体进行了重新研究，以确定该实验系统中基因内互补的分子解释。在对磷酸酶阴性的phoA基因进行PCR扩增后，通过DNA序列分析确定这些突变的位置。大多数突变涉及金属结合位点的配体。通过定点诱变重新创建每个突变酶，进行表达、纯化并进行动力学表征。为了研究两个亚基之间的协同作用，我们分析了一些定点突变酶的异二聚体形式。为了能够以纯形式分离异二聚体碱性磷酸酶，通过用三个天冬氨酸残基取代C末端赖氨酸残基来改变一个亚基的总电荷。这种修饰对酶的动力学性质没有影响。使用两种方法产生异二聚体碱性磷酸酶：（1）在酸性pH下解离，然后在锌和镁离子存在下于略碱性pH条件下重新缔合，在体外形成；（2）从携带两个不同phoA基因的质粒在体内表达。对于某些突变酶组合，观察到k(cat)增加以及对硝基苯磷酸酯K(m)大幅降低。这些结果表明，大肠杆菌碱性磷酸酶组装成二聚体诱导了单体之间的协同相互作用，这对于全酶功能形式的形成是必需的。
