Collins J C, Permuth S F, Brooker R J
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
J Biol Chem. 1989 Sep 5;264(25):14698-703.
In the present study, lactose permease mutants were isolated which have an enhanced recognition toward maltose (an alpha-glucoside) and diminished recognition for cellobiose (a beta-glucoside). Nine mutants were isolated from a strain encoding a wild-type permease (pTE18) and nine from a strain encoding a mutant permease which recognizes maltose (pB15). All 18 mutants were subjected to DNA sequencing, and it was found that all mutations are single base substitutions within the lac Y gene effecting single amino acid substitutions within the protein. From the pTE18 parent, substitutions involved Tyr-236 to Phe or His; Ser-306 to Thr; and six independent mutants in which Ala-389 was changed to Pro. From pB15, Tyr-236 was changed to Phe or Asn, Ser-306 to Thr or Leu, Lys-319 to Asn, and His-322 to Tyr, Asn, or Gln. All 18 mutants exhibited enhanced recognition for maltose (compared with the pTE18 strain) and a diminished recognition for cellobiose. In addition, all mutants showed a diminished recognition toward beta-galactosides as well. The Phe-236, His-236, Leu-306, Asn-319, Tyr-322, Asn-322, and Gln-322 mutants were completely defective in the uphill accumulation of methyl-beta-D-thiogalactopyranoside whereas the Asn-236, Thr-306, and Pro-389 mutants could effectively accumulate methyl-beta-D-thiogalactopyranoside against a concentration gradient. The mutants obtained in this study, together with previous lactose permease mutants, tend to be found on transmembrane segments, and those which are on the same transmembrane segment are often found three or four amino acids away from each other. This pattern is consistent with a protein structure in which important amino acid side chains project from several transmembrane segments in such a way as to form a hydrophilic channel for the recognition and transport of H+ and galactosides. It is proposed that the mechanism for H+/lactose cotransport is consistent with a "flanking gate" model in which the protein contains a single recognition site for galactosides within the channel which is flanked on either side by gates.
在本研究中，分离出了乳糖通透酶突变体，它们对麦芽糖（一种α-葡萄糖苷）的识别增强，而对纤维二糖（一种β-葡萄糖苷）的识别减弱。从编码野生型通透酶的菌株（pTE18）中分离出9个突变体，从编码识别麦芽糖的突变通透酶的菌株（pB15）中分离出9个突变体。对所有18个突变体进行了DNA测序，发现所有突变都是lac Y基因内的单碱基替换，导致蛋白质内的单个氨基酸替换。来自pTE18亲本的替换包括Tyr-236替换为Phe或His；Ser-306替换为Thr；以及6个独立的突变体，其中Ala-389变为Pro。来自pB15的突变包括Tyr-236变为Phe或Asn，Ser-306变为Thr或Leu，Lys-319变为Asn，His-322变为Tyr、Asn或Gln。所有18个突变体对麦芽糖的识别增强（与pTE18菌株相比），对纤维二糖的识别减弱。此外，所有突变体对β-半乳糖苷的识别也减弱。Phe-236、His-236、Leu-306、Asn-319、Tyr-322、Asn-322和Gln-322突变体在甲基-β-D-硫代半乳糖吡喃糖苷的上坡积累方面完全缺陷，而Asn-236、Thr-306和Pro-389突变体能够有效地逆浓度梯度积累甲基-β-D-硫代半乳糖吡喃糖苷。本研究中获得的突变体与先前的乳糖通透酶突变体一样，倾向于出现在跨膜区段上，并且位于同一跨膜区段上的突变体通常彼此相隔三或四个氨基酸。这种模式与一种蛋白质结构一致，在该结构中，重要的氨基酸侧链从几个跨膜区段伸出，以形成一个用于识别和运输H⁺和半乳糖苷的亲水通道。有人提出，H⁺/乳糖共转运的机制与“侧翼门”模型一致。在该模型中，蛋白质在通道内含有一个半乳糖苷的单一识别位点，其两侧各有一个门。
