Sauna Zuben E, Müller Marianna, Peng Xiang-Hong, Ambudkar Suresh V
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4254, USA.
Biochemistry. 2002 Nov 26;41(47):13989-4000. doi: 10.1021/bi026626e.
The human MDR1 (ABCB1) gene product, P-glycoprotein (Pgp), functions as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. In this study, we assessed the role of conserved glutamate residues in the Walker B domain of the two ATP sites (E556 and E1201, respectively) during the catalytic cycle of human Pgp. The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [alpha-(32)P]-8-azidoADP was trapped in the presence of vanadate (Vi), and the release of trapped [alpha-(32)P]-8-azidoADP occurred to a similar extent as in wild-type Pgp. This indicates that these mutations do not affect either the first hydrolysis event or the ADP release step. Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Following the first hydrolysis event and release of [alpha-(32)P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [alpha-(32)P]-8-azidoADP trapping. Interestingly, the double mutants E556/1201Q and E556/1201A trapped [alpha-(32)P]-8-azidoADP even in the absence of Vi, and the occluded nucleotide was not released after incubation at 37 degrees C for an extended period. In addition, the properties of transition state conformation of the double mutants generated in the absence of Vi were found to be similar to that of the wild-type protein trapped in the presence of Vi (Pgp x [alpha-(32)P]-8-azidoADP xVi). Thus, in contrast to the single mutants, the double mutants appear to be defective in the ADP release step. In aggregate, these data suggest that E556 and E1201 residues in the Walker B domains may not be critical as catalytic carboxylates for the cleavage of the bond between the gamma-P and the beta-P of ATP during hydrolysis but are essential for the second ATP hydrolysis step and completion of the catalytic cycle.
人类多药耐药基因1（MDR1，即ABCB1）的基因产物P-糖蛋白（Pgp）作为一种ATP依赖的外排泵，可转运多种化疗药物。在本研究中，我们评估了人Pgp催化循环过程中，两个ATP位点的沃克B结构域中保守谷氨酸残基（分别为E556和E1201）的作用。使用基于痘苗病毒的表达系统对突变型Pgp（E556Q、E556A、E1201Q、E1201A、E556/1201Q和E556/1201A）进行了表征。虽然E556Q和E1201Q突变型Pgp的稳态ATP水解和药物转运活性均被消除，但在钒酸盐（Vi）存在的情况下，[α-(32)P]-8-叠氮基ADP被捕获，且捕获的[α-(32)P]-8-叠氮基ADP的释放程度与野生型Pgp相似。这表明这些突变既不影响第一次水解事件，也不影响ADP释放步骤。当谷氨酸残基被丙氨酸取代（E556A和E1201A）时，也获得了类似的结果。在第一次水解事件以及[α-(32)P]-8-叠氮基ADP释放后，E556Q和E1201Q突变型Pgp均未能经历Vi诱导的[α-(32)P]-8-叠氮基ADP捕获的另一个循环。有趣的是，双突变体E556/1201Q和E556/1201A即使在没有Vi的情况下也能捕获[α-(32)P]-8-叠氮基ADP，并且在37℃下长时间孵育后，封闭的核苷酸也不会释放。此外，发现在没有Vi的情况下产生的双突变体的过渡态构象性质与在有Vi的情况下捕获的野生型蛋白（Pgp x [α-(32)P]-8-叠氮基ADP xVi）相似。因此，与单突变体不同，双突变体似乎在ADP释放步骤中存在缺陷。总的来说，这些数据表明，沃克B结构域中的E556和E1201残基在水解过程中作为催化羧酸盐对于ATP的γ-P和β-P之间的键的断裂可能并不关键，但对于第二个ATP水解步骤和催化循环的完成是必不可少的。
