Davidson A L, Laghaeian S S, Mannering D E
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 1996 Mar 1;271(9):4858-63.
Maltose transport across the cytoplasmic membrane of Escherichia coli is catalyzed by a periplasmic binding protein-dependent transport system and energized by ATP. The maltose system, a member of the ATP-binding cassette or ABC transport family, contains two copies of an ATP-binding protein in a complex with two integral membrane proteins. ATP hydrolysis by the transport complex can be assayed following reconstitution into proteoliposomes in the presence of maltose binding protein and maltose. Mutations in the transport complex that permit binding protein-independent transport render ATP hydrolysis constitutive so that hydrolysis can also be assayed with the transport complex in detergent solution. We have used both of these systems to study the role of two ATP binding sites in ATP hydrolysis. We found that both the wild-type and the binding protein-independent systems hydrolyzed ATP with positive cooperativity, suggesting that the two ATP binding sites interact. Vanadate inhibited the ATPase activity of the transport complex with 50% inhibition occurring at 10 mum vanadate. In detergent solution, the degree of cooperativity in the binding protein-independent complex decreased with increasing pH. The loss of cooperativity was accompanied by a decrease in ATPase activity and a decrease in sensitivity to vanadate. Because reconstitution of the complex into a lipid bilayer prevented the loss of cooperativity, we expect that ATP hydrolysis is cooperative in vivo. The mutations leading to binding protein-independent transport do not significantly alter the affinity, cooperativity, vanadate sensitivity, or substrate specificity of the ATP binding sites during hydrolysis. These results justify the use of the binding protein-independent system to investigate the mechanism of transport and hydrolysis.
麦芽糖跨大肠杆菌细胞质膜的转运由一种依赖周质结合蛋白的转运系统催化，并由ATP提供能量。麦芽糖系统是ATP结合盒（ABC）转运家族的成员，它包含两个ATP结合蛋白拷贝，与两个整合膜蛋白形成复合物。在麦芽糖结合蛋白和麦芽糖存在的情况下，将转运复合物重组到蛋白脂质体中后，可以检测转运复合物对ATP的水解作用。转运复合物中允许不依赖结合蛋白进行转运的突变会使ATP水解组成型，因此也可以在去污剂溶液中用转运复合物检测水解作用。我们使用这两种系统来研究两个ATP结合位点在ATP水解中的作用。我们发现，野生型和不依赖结合蛋白的系统都以正协同性水解ATP，这表明两个ATP结合位点相互作用。钒酸盐抑制转运复合物的ATP酶活性，在10 μM钒酸盐时抑制率达到50%。在去污剂溶液中，不依赖结合蛋白的复合物中的协同程度随pH值升高而降低。协同性的丧失伴随着ATP酶活性的降低和对钒酸盐敏感性的降低。由于将复合物重组到脂质双层中可防止协同性丧失，我们预计ATP水解在体内是协同进行的。导致不依赖结合蛋白进行转运的突变在水解过程中不会显著改变ATP结合位点的亲和力、协同性、钒酸盐敏感性或底物特异性。这些结果证明使用不依赖结合蛋白的系统来研究转运和水解机制是合理的。
