Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
Biochemistry. 2014 Feb 18;53(6):991-1000. doi: 10.1021/bi401280v. Epub 2014 Feb 7.
P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug-drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible. With γ(18)O4-labeled ATP, no positional isotope exchange is detectable at the bridging β-phosphorus-O-γ-phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three (18)O/two (18)O/one (18)O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO4(2-) (Pi) was negligible, suggesting that a [P-gp·ADP·Pi] is not long-lived. This further demonstrates that the hydrolysis is essentially irreversible in the active site. These mechanistic details of ATP hydrolysis are consistent with a very fast conformational change immediately following, or concomitant with, hydrolysis of the γ-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states.
P-糖蛋白（P-gp）是 ABC 转运蛋白家族的一员，通过催化其外排作用使许多肿瘤产生耐药性，是药物相互作用的主要组成部分。P-gp 通过协调药物结合部位的构象变化与 ATP 的水解和 ADP 的释放，将药物外排与 ATP 水解偶联。为了了解水解的化学步骤和随后的构象变化的相对速率，我们利用同位素交换方法来确定 ATP 水解步骤的可逆程度。使用 γ(18)O4 标记的 ATP，在桥接β-磷酸-O-γ-磷酸键处没有可检测到的位置同位素交换。此外，水解产生的磷酸盐包括一个恒定的三（18）O/两（18）O/一（18）O 比值，这反映了多个实验中起始 ATP 的同位素组成。因此，H2O 与 HPO4(2-)（Pi）的交换可以忽略不计，这表明 [P-gp·ADP·Pi] 不会长期存在。这进一步证明了水解在活性位点基本上是不可逆的。这些 ATP 水解的机制细节与紧随其后或同时发生的γ-磷酸键的快速构象变化一致，这确保了在无药物和药物结合状态下对催化作用的高度承诺。
