Carrasco N, Püttner I B, Antes L M, Lee J A, Larigan J D, Lolkema J S, Roepe P D, Kaback H R
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Biochemistry. 1989 Mar 21;28(6):2533-9. doi: 10.1021/bi00432a028.
lac permease with Ala in place of Glu325 was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The reconstituted molecule is completely unable to catalyze lactose/H+ symport but catalyzes exchange and counterflow at least as well as wild-type permease. In addition, Ala325 permease catalyzes downhill lactose influx without concomitant H+ translocation and binds p-nitrophenyl alpha-D-galactopyranoside with a KD only slightly higher than that of wild-type permease. Studies with right-side-out membrane vesicles demonstrate that replacement of Glu325 with Gln, His, Val, Cys, or Trp results in behavior similar to that observed with Ala in place of Glu325. On the other hand, permease with Asp in place of Glu325 catalyzes lactose/H+ symport about 20% as well as wild-type permease. The results indicate that an acidic residue at position 325 is essential for lactose/H+ symport and that hydrogen bonding at this position is insufficient. Taken together with previous results and those presented in the following paper [Lee, J. A., Püttner, I. B., & Kaback, H. R. (1989) Biochemistry (third paper of three in this issue)], the findings are consistent with the idea that Arg302, His322, and Glu325 may be components of a H+ relay system that plays an important role in the coupled translocation of lactose and H+.
将膜中用丙氨酸取代谷氨酸325的乳糖通透酶溶解、纯化并重新组装到蛋白脂质体中。重新组装的分子完全无法催化乳糖/H⁺同向转运，但催化交换和逆向流动的能力至少与野生型通透酶一样好。此外，丙氨酸325通透酶催化乳糖的顺浓度梯度内流，且不伴随H⁺转运，并且与对硝基苯基α-D-吡喃半乳糖苷结合，其解离常数仅略高于野生型通透酶。对外翻膜囊泡的研究表明，用谷氨酰胺、组氨酸、缬氨酸、半胱氨酸或色氨酸取代谷氨酸325会导致与用丙氨酸取代谷氨酸325时观察到的行为相似。另一方面，用天冬氨酸取代谷氨酸325的通透酶催化乳糖/H⁺同向转运的能力约为野生型通透酶的20%。结果表明，325位的酸性残基对于乳糖/H⁺同向转运至关重要，并且该位置处的氢键作用并不充分结合先前的结果以及以下论文[Lee,J.A.,Püttner,I.B.,&Kaback,H.R.(1989)Biochemistry(本期三篇论文中的第三篇)]中给出的数据，这些发现与精氨酸302、组氨酸322和谷氨酸325可能是H⁺传递系统的组成部分这一观点一致，该系统在乳糖和H⁺的偶联转运中起重要作用。
