Gutknecht R, Manni M, Mao Q, Erni B
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
J Biol Chem. 1998 Oct 2;273(40):25745-50. doi: 10.1074/jbc.273.40.25745.
The bacterial phosphotransferase system (PTS) consists of two energy-coupling soluble proteins (enzyme I and HPr) and a large number of inner membrane transporters (enzymes II) that mediate concomitant phosphorylation and translocation of sugars and hexitols. The transporters consist of three functional units (IIA, IIB, IIC), which occur either as protein subunits or domains of a multidomain polypeptide. The membrane-spanning IIC domain contains the substrate binding site; IIA and IIB are phosphorylation domains that transfer phosphate from HPr to the transported sugar. The transporter complexes of the PTS are good examples for variation of design by modular assembly of domains and subunits. The domain order is IIC-IIB in the membrane subunit of the Escherichia coli glucose transporter (IICBGlc) and IIB-IIC in Salmonella typhimurium sucrose transporter (IIBCScr). The phosphorylation domain of IICBGlc was translocated from the carboxyl-terminal to the amino-terminal end of the IIC domain, and the activity of the circularly permuted form was optimized by variation of the length and the composition of the interdomain linker. IIBapCGlc with an alanine-proline-rich interdomain linker has 70% of the control specific activity after purification and reconstitution into proteoliposomes. These results indicate that the amino-terminal end of IICBGlc must be on the cytoplasmic side of the inner membrane, that membrane insertion of the IIC domain is insensitive to the modification of its amino-terminal end, and that a domain swap as it could occur by a single DNA translocation event can rapidly lead to a functional protein. However, IIB could not be substituted for by glucokinase. Fusion proteins between the IIC domain and glucokinase do not transport and phosphorylate glucose in an ATP-dependent mechanism, although the IIC moiety displays transport activity upon complementation with soluble subclonal IIB, and the glucokinase moiety retains ATP-dependent nonvectorial kinase activity. This indicates that IIC and IIB are two cooperative units and not only sequentially acting upon a common substrate, and that translocation of glucose must be conformationally coupled to the phosphorylation/dephosphorylation cycle of IIB.
细菌磷酸转移酶系统（PTS）由两种能量偶联可溶性蛋白（酶I和HPr）以及大量内膜转运蛋白（酶II）组成，这些转运蛋白介导糖类和己糖醇的磷酸化与跨膜转运过程同步进行。转运蛋白由三个功能单元（IIA、IIB、IIC）组成，它们可以作为蛋白质亚基或多结构域多肽的结构域存在。跨膜的IIC结构域包含底物结合位点；IIA和IIB是磷酸化结构域，负责将磷酸基团从HPr转移至被转运的糖类上。PTS的转运蛋白复合物是通过结构域和亚基的模块化组装实现设计多样化的典型例子。在大肠杆菌葡萄糖转运蛋白（IICBGlc）的膜亚基中，结构域顺序为IIC-IIB，而在鼠伤寒沙门氏菌蔗糖转运蛋白（IIBCScr）中则为IIB-IIC。IICBGlc的磷酸化结构域从IIC结构域的羧基末端转移至氨基末端，通过改变结构域间连接子的长度和组成，优化了环形排列形式的活性。带有富含丙氨酸-脯氨酸连接子的IIBapCGlc在纯化并重组到蛋白脂质体后，具有对照比活性的70%。这些结果表明，IICBGlc的氨基末端必须位于内膜的细胞质一侧，IIC结构域插入膜中对其氨基末端的修饰不敏感，并且单个DNA易位事件可能发生的结构域交换能够迅速产生功能性蛋白。然而，IIB不能被葡萄糖激酶替代。IIC结构域与葡萄糖激酶之间的融合蛋白不能以ATP依赖机制转运和磷酸化葡萄糖，尽管IIC部分在与可溶性亚克隆IIB互补时表现出转运活性，并且葡萄糖激酶部分保留了ATP依赖的非载体激酶活性。这表明IIC和IIB是两个协同单元，不仅依次作用于共同底物，而且葡萄糖的转运必须在构象上与IIB的磷酸化/去磷酸化循环偶联。
