ROLE OF GLYCINE RESIDUES IN THE STRUCTURE AND FUNCTION OF LACTOSE PERMEASE, AN ESCHERICHIA-COLI MEMBRANE-TRANSPORT PROTEIN
ROLE OF GLYCINE RESIDUES IN THE STRUCTURE AND FUNCTION OF LACTOSE PERMEASE, AN ESCHERICHIA-COLI MEMBRANE-TRANSPORT PROTEIN
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DOI：
10.1021/bi00003a038
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发表时间：
1995-01-24
期刊：
影响因子：
2.9
通讯作者：
KABACK, HR
中科院分区：
文献类型：
--
作者：
JUNG, K;JUNG, H;KABACK, HR
采用寡核苷酸定向、位点特异性诱变的方法，系统地研究了34个Gly残基在大肠杆菌乳糖渗透酶中的作用。在一个缺乏Cys残基的功能性渗透酶突变体（无c -渗透酶）中，36个Gly残基中的34个残基都被Cys取代，正如先前的实验表明，Gly-402和Gly-404可以通过截断c端而不丧失活性而被删除[Roepe， P. D., et al. (1989) Proc. Natl.]。学会科学。美国86,3992；麦肯纳等人（1991）。学会科学。[美]。在所描述的34个Cys替代突变体中，15个具有高活性的乳糖转运，16个表现出较低但显著的乳糖催化积累能力，3个（Gly-64- >Cys, Gly-115- >Cys和Gly-147- >Cys）没有任何活性。用Ala、Val或Pro代替Gly对失活突变体进行了更详细的研究。含有Gly-115- >Ala或Gly-147- >Ala的无c渗透酶几乎可以像对照组一样运输乳糖，而含有Val或Pro代替Gly的突变体很少或没有积累双糖的能力。相反，用Ala、Val或Pro代替Gly-64的突变体是无活性的。然而，引人注目的是，当突变被放在野生型背景下时，gy -64- >Ala渗透酶转运乳糖，β， d-半乳糖酰氨基乙酯1-硫- β， d-半乳糖酰氨基乙酯和甲基1-硫- β， d-半乳糖酰氨基乙酯40-60%以及野生型渗透酶，而gy -64- >Val或gy -64- >Pro渗透酶对所有这些底物都无活性。结果表明，虽然乳糖渗透酶中的Gly残基对活性没有强制要求，但64、115和147位侧链的体积，而不是这些位置的构象灵活性，是特别重要的。
By using oligonucleotide-directed, site-specific mutagenesis, the role of 34 Gly residues in the lactose permease of Escherichia coli has been studied systematically. Each of 34 out of a total of 36 Gly residues was replaced with Cys in a functional permease mutant devoid of Cys residues (C-less permease), as previous experiments demonstrate that Gly-402 and Gly-404 can be deleted by truncation of the C-terminus with no loss of activity [Roepe, P. D., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3992; McKenna, E., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969]. Out of the 34 Cys-replacement mutants described, 15 transport lactose with high activity, 16 exhibit decreased but significant ability to catalyze lactose accumulation, and 3 (Gly-64-->Cys, Gly-115-->Cys and Gly-147-->Cys) exhibit no activity whatsoever. The inactive mutants were studied in more detail by replacement of Gly with Ala, Val, or Pro. C-less permease with Gly-115-->Ala or Gly-147-->Ala transports lactose almost as well as the control, while mutants with Val or Pro in place of Gly have little or no capacity to accumulate the disaccharide. In contrast, mutants with Ala, Val, or Pro in place of Gly-64 are inactive. Strikingly, however, when the mutations are placed in the wild-type background, Gly-64-->Ala permease transports lactose, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside, and methyl 1-thio-beta,D-galactopyranoside 40-60% as well as wild-type permease, while Gly-64-->Val or Gly-64-->Pro permease is inactive toward all of these substrates. The results indicate that although none of the Gly residues in lactose permease is mandatory for activity, the bulk of the side chain at positions 64, 115, and 147, rather than conformational flexibility at these positions, is particularly important.