Franco P J, Brooker R J
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
J Biol Chem. 1991 Apr 15;266(11):6693-9.
The single asparagine 322 mutant of the lactose permease was made by constructing a hybrid plasmid which contained the amino-terminal coding sequence from the wild-type permease gene and the carboxyl-terminal coding sequence from a previously characterized double mutant permease which contained an asparagine residue at position 322. Since histidine at position 322 has been postulated to be critically involved with H+ transport and the active accumulation of sugars, the ability of the Asn-322 mutant to couple H+ and sugar transport was carefully examined. Measurements of proton/lactose stoichiometries gave very similar values for the wild-type (0.78) and the Asn-322 strain (0.82). Moreover, the Asn-322 mutant was able to effectively accumulate lactose against a concentration gradient although the levels of accumulation in the Asn-322 mutant (approximately 5-7-fold) were significantly less than that of the wild-type strain (approximately 30-40-fold). Overall, these results are inconsistent with the notion that an ionizable histidine residue at position 322 is obligatorily required for H+ transport or the active accumulation of galactosides against a concentration gradient. The ability of the Asn-322 mutant to recognize a variety of sugars was compared with wild-type, Val-177, and Val-177/Asn-322 strains. The Asn-322 mutant exhibited an ability to recognize and transport maltose (an alpha-glucoside) which was significantly better than the wild-type strain but not as good as either the single Val-177 mutant or the double Val-177/Asn-322 mutant. Both the Asn-322 and the Val-177/Asn-322 strain showed a relatively poor recognition for alpha-galactosides (i.e. melibiose), beta-galactosides (lactose and thiodigalactoside), and beta-glucosides (cellobiose). In contrast, the single Val-177 strain exhibited a normal recognition for these sugars.
乳糖通透酶的单个天冬酰胺322突变体是通过构建一个杂交质粒而获得的，该质粒含有野生型通透酶基因的氨基末端编码序列和一个先前已鉴定的双突变通透酶的羧基末端编码序列，该双突变通透酶在322位含有一个天冬酰胺残基。由于322位的组氨酸被推测与H⁺转运和糖的主动积累密切相关，因此对Asn - 322突变体偶联H⁺和糖转运的能力进行了仔细研究。质子/乳糖化学计量比的测量结果显示，野生型（0.78）和Asn - 322菌株（0.82）的值非常相似。此外，Asn - 322突变体能够有效地逆浓度梯度积累乳糖，尽管Asn - 322突变体中的积累水平（约5 - 7倍）明显低于野生型菌株（约30 - 40倍）。总体而言，这些结果与以下观点不一致，即322位的可电离组氨酸残基是H⁺转运或半乳糖苷逆浓度梯度主动积累所必需的。将Asn - 322突变体识别多种糖的能力与野生型、Val - 177和Val - 177/Asn - 322菌株进行了比较。Asn - 322突变体表现出识别和转运麦芽糖（一种α - 葡萄糖苷）的能力，该能力明显优于野生型菌株，但不如单个Val - 177突变体或双Val - 177/Asn - 322突变体。Asn - 322和Val - 177/Asn - 322菌株对α - 半乳糖苷（即蜜二糖）、β - 半乳糖苷（乳糖和硫代二半乳糖苷）和β - 葡萄糖苷（纤维二糖）的识别相对较差。相比之下，单个Val - 177菌株对这些糖表现出正常的识别能力。
