Reaction Dynamics of ATP Hydrolysis Catalyzed by P-Glycoprotein

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• 作者：Michele Scian ; Mauro Acchione ; Mavis Li ; William M. Atkins
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：February 18, 2014
• 年：2014
• 卷：53
• 期：6
• 页码：991-1000
• 全文大小：526K
• ISSN：1520-4995

文摘

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鈥揹rug 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 纬¹⁸O₄-labeled ATP, no positional isotope exchange is detectable at the bridging 尾-phosphorus鈥揙鈭捨?phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three ¹⁸O/two ¹⁸O/one ¹⁸O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H₂O-exchange with HPO₄^{2鈥?/sup> (P_i) was negligible, suggesting that a [P-gp路ADP路P_i] 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 纬-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states.}