Goswitz V C, Brooker R J
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
Membr Biochem. 1993 Jan-Mar;10(1):61-70. doi: 10.3109/09687689309150253.
In the present study lactose permease mutants were isolated which recognize the monosaccharide, L-arabinose. Although the wild-type permease exhibits a poor recognition for L-arabinose, seven independent mutants were identified by their ability to grow on L-arabinose minimal plates. When subjected to DNA sequencing, it was found that all seven of these mutants were single-site mutations in which alanine 177 was changed to valine. The wild type and valine 177 mutant were then analyzed with regard to their abilities to recognize and transport monosaccharides and disaccharides. Free L-arabinose was shown to competitively inhibit [14C]-lactose transport yielding a Ki value of 121 mM for the Val177 mutant and a much higher value of 320 mM for the wild-type. Among several monosaccharides, D-glucose as well as L-arabinose inhibited lactose transport in the Val177 mutant to a significantly greater extent, while D-arabinose and D-xylose only caused a slight inhibition. On the other hand, kinetic studies with sugars which are normally recognized by the wild-type permease such as [14C]-galactose and [14C]-lactose revealed that the Val177 mutant and wild-type strains had similar transport characteristics for these two sugars. Overall, these results are consistent with the notion that the Val177 substitution causes an enhanced recognition for particular sugars (i.e. L-arabinose) but does not universally affect the recognition and unidirectional transport for all sugars. This idea is further supported by the observation that site-directed mutants containing isoleucine, leucine, phenylalanine, or proline at position 177 also were found to possess an enhanced recognition for L-arabinose.
在本研究中，分离出了能识别单糖L-阿拉伯糖的乳糖通透酶突变体。尽管野生型通透酶对L-阿拉伯糖的识别能力较差，但通过其在L-阿拉伯糖基本平板上生长的能力鉴定出了7个独立的突变体。对其进行DNA测序时发现，所有这7个突变体均为单点突变，其中丙氨酸177被替换为缬氨酸。然后分析了野生型和缬氨酸177突变体识别和转运单糖及双糖的能力。结果表明，游离L-阿拉伯糖竞争性抑制[14C]-乳糖转运，缬氨酸177突变体的Ki值为121 mM，而野生型的Ki值则高得多，为320 mM。在几种单糖中，D-葡萄糖和L-阿拉伯糖对缬氨酸177突变体中乳糖转运的抑制作用明显更强，而D-阿拉伯糖和D-木糖仅引起轻微抑制。另一方面，对通常被野生型通透酶识别的糖类（如[14C]-半乳糖和[14C]-乳糖）进行动力学研究发现，缬氨酸177突变体和野生型菌株对这两种糖具有相似的转运特性。总体而言，这些结果与以下观点一致：缬氨酸177替换导致对特定糖类（即L-阿拉伯糖）的识别增强，但并非普遍影响对所有糖类的识别和单向转运。在第177位含有异亮氨酸、亮氨酸、苯丙氨酸或脯氨酸的定点突变体也被发现对L-阿拉伯糖具有增强的识别能力，这一观察结果进一步支持了这一观点。
