Johnson J L, Brooker R J
Department of Genetics and Cell Biology and the Institute for Advanced Studies in Biological Process Technology, University of Minnesota, St. Paul, Minnesota 55108, USA.
J Biol Chem. 1999 Feb 12;274(7):4074-81. doi: 10.1074/jbc.274.7.4074.
In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains. The wild-type and Asn-319 mutant showed high levels of lactose uptake, with Km values of 0.42 and 1.30 mM and Vmax values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively. The Asn-319/Gln-325 strain had a normal Km of 0.36 mM and a moderate Vmax of 18.5 nmol of lactose/min/mg of protein. By comparison, the single E325Q strain had a normal Km of 0.27 mM but a very defective Vmax of 1.3 nmol of lactose/min/mg of protein. A similar trend was observed among the alanine substitutions at these positions, although the Vmax values were lower for the Ala-319 mutations. When comparing the Vmax values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in Vmax caused by neutral 325 mutations. With regard to H+/lactose coupling, the wild-type permease is normally coupled and can transport lactose against a gradient. The position 325 single mutants showed no evidence of H+ transport with lactose or thiodigalactoside (TDG) and were unable to facilitate uphill lactose transport. The single Asn-319 mutant and double Asn-319/Gln-325 mutant were able to transport H+ upon the addition of lactose or TDG. In addition, both of these strains catalyzed a sugar-dependent H+ leak that inhibited cell growth in the presence of TDG. These two strains were also defective in uphill transport, which may be related to their sugar-dependent leak pathway. Based on these and other results in the literature, a model is presented that describes how the interactions among several ionizable residues within the lactose permease act in a concerted manner to control H+/lactose coupling. In this model, Lys-319 and Glu-325 play a central role in governing the ability of the lactose permease to couple the transport of H+ and lactose.
在本研究中，我们检测了野生型乳糖通透酶、赖氨酸-319被替换为天冬酰胺或丙氨酸以及谷氨酸-325被替换为谷氨酰胺或丙氨酸的单突变体，以及相应的双突变体菌株的转运特性。野生型和天冬酰胺-319突变体表现出高水平的乳糖摄取，Km值分别为0.42和1.30 mM，Vmax值分别为102.6和48.3 nmol乳糖/分钟/毫克蛋白质。天冬酰胺-319/谷氨酰胺-325菌株的正常Km为0.36 mM，中等Vmax为18.5 nmol乳糖/分钟/毫克蛋白质。相比之下，单E325Q菌株的正常Km为0.27 mM，但Vmax非常低，为1.3 nmol乳糖/分钟/毫克蛋白质。在这些位置的丙氨酸替换中也观察到类似趋势，尽管丙氨酸-319突变的Vmax值较低。当比较325位单突变体与双突变体的Vmax值时，这些结果表明319位的中性突变显著减轻了325位中性突变导致的Vmax缺陷。关于H⁺/乳糖偶联，野生型通透酶正常偶联，能够逆梯度转运乳糖。325位单突变体未显示出与乳糖或硫代二半乳糖苷（TDG）进行H⁺转运的证据，并且无法促进乳糖的上坡转运。单天冬酰胺-319突变体和双天冬酰胺-319/谷氨酰胺-325突变体在添加乳糖或TDG后能够转运H⁺。此外，这两种菌株都催化了一种糖依赖性H⁺泄漏，在存在TDG的情况下抑制细胞生长。这两种菌株在上坡转运方面也存在缺陷，这可能与其糖依赖性泄漏途径有关。基于这些以及文献中的其他结果，提出了一个模型，该模型描述了乳糖通透酶内几个可电离残基之间的相互作用如何协同作用以控制H⁺/乳糖偶联。在这个模型中，赖氨酸-319和谷氨酸-325在控制乳糖通透酶偶联H⁺和乳糖转运的能力方面起着核心作用。
