Sutrina S L, Reddy P, Saier M H, Reizer J
Department of Biology, University of California at San Diego, La Jolla 92093.
J Biol Chem. 1990 Oct 25;265(30):18581-9.
Biochemical, immunological, and sequence analyses demonstrated that the glucose permease of Bacillus subtilis, the glucose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system, is a single polypeptide chain with a C-terminal Enzyme III-like domain. A flexible hydrophilic linker, similar in length and amino acid composition to linkers previously identified in other regulatory or sensory transducing proteins, functions to tether the Enzyme IIIGlc-like domain of the protein to the membrane-embedded Enzyme IIGlc. Evidence is presented demonstrating that the Enzyme IIIGlc-like domain of the glucose permease plays a dual role and functions in the transport and phosphorylation of both glucose and sucrose. The sucrose permease appears to lack a sucrose-specific Enzyme III-like domain or a separate, soluble IIIScr protein. Enzyme IIScr was capable of utilizing the IIIGlc-like domain of the glucose permease regardless of whether the IIIGlc polypeptide was provided as a purified, soluble protein, as a membrane-bound protein within the same membrane as Enzyme IIScr, or as a membrane-bound protein within membrane fragments different from those bearing Enzyme IIScr. These observations suggest that the IIIGlc-like domain is an autonomous structural unit that assumes a conformation independent of the hydrophobic, N-terminal intramembranal domain of Enzyme IIGlc. Preferential uptake and phosphorylation of glucose over sucrose has been demonstrated by both in vivo transport studies and in vitro phosphorylation assays. Addition of the purified IIIGlc-like domain strongly stimulated the phosphorylation of sucrose, but not that of glucose, in phosphorylation assays that contained the two sugars simultaneously. The results suggest that the preferential uptake of glucose over sucrose is determined by competition of the corresponding sugar-specific permeases for the common P approximately IIIGlc/Scr domain.
生化、免疫和序列分析表明，枯草芽孢杆菌的葡萄糖通透酶，即磷酸烯醇丙酮酸依赖性磷酸转移酶系统中葡萄糖特异性的酶II，是一条具有C末端类酶III结构域的单多肽链。一个灵活的亲水性连接子，其长度和氨基酸组成与先前在其他调节或传感转导蛋白中鉴定出的连接子相似，其作用是将该蛋白的类酶IIIGlc结构域与膜嵌入的酶IIGlc相连。有证据表明，葡萄糖通透酶的类酶IIIGlc结构域具有双重作用，在葡萄糖和蔗糖的转运及磷酸化过程中均发挥功能。蔗糖通透酶似乎缺乏蔗糖特异性的类酶III结构域或单独的可溶性IIIScr蛋白。无论类IIIGlc多肽是以纯化的可溶性蛋白形式、作为与酶IIScr在同一膜中的膜结合蛋白形式，还是作为与携带酶IIScr的膜片段不同的膜片段中的膜结合蛋白形式提供，酶IIScr都能够利用葡萄糖通透酶的类IIIGlc结构域。这些观察结果表明，类IIIGlc结构域是一个自主的结构单元，其构象独立于酶IIGlc的疏水N末端膜内结构域。体内转运研究和体外磷酸化分析均已证明，相较于蔗糖，葡萄糖具有优先摄取和磷酸化的特性。在同时含有两种糖的磷酸化分析中，添加纯化的类IIIGlc结构域强烈刺激了蔗糖的磷酸化，但对葡萄糖的磷酸化没有影响。结果表明，葡萄糖相较于蔗糖的优先摄取是由相应糖特异性通透酶对共同的P≈IIIGlc/Scr结构域的竞争所决定的。
