Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein
Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein
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DOI：
10.1021/bi034257w
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发表时间：
2003-11-11
期刊：
影响因子：
2.9
通讯作者：
Gros, P
中科院分区：
文献类型：
--
作者：
Carrier, I;Julien, M;Gros, P
在 ABC 转运蛋白的核苷酸结合域 (NBD) 中，例如小鼠 Mdr3 P-糖蛋白 (P-gp)，不变的羧酸残基（NBD1 中的 E552；NBD2 中的 E1197）紧随 Walker B 基序 (hyd(4)DE/D)。突变体 E552Q 和 E1197Q 中负电荷的去除消除了通过 P-i 释放测量的药物刺激的 ATP 酶活性。令人惊讶的是，在过渡态类似物钒酸盐 (V-i) 存在和不存在的情况下，在突变体中仍然观察到药物刺激的 8-叠氮基-[α-P-32]ATP 捕获，并且可以从捕获的酶中回收 ADP。 E552Q 和 E1197Q 突变体在 8-叠氮基-[α-P-32]ATP 结合和 8-叠氮基-[α-P-32]核苷酸捕获方面表现出与野生型 (WT) 酶相似的特征，后者同时依赖于 Mg2+ 和温度。重要的是，E552Q 中药物刺激的核苷酸捕获受 V-i 刺激并且类似于 WT 酶，而 E1197Q 中它几乎完全对 V-i 不敏感。当氟化铝或氟化铍用作替代过渡态类似物时，观察到类似的核苷酸捕获特性。光标记酶的部分蛋白水解裂解表明，在不存在 V-i 的情况下，核苷酸捕获仅发生在突变体 NBD 处，而在存在 V-i 的情况下，核苷酸捕获发生在两个 NBD 处。总之，这些结果表明，E552Q 和 E1197Q 突变体中发生单位点转换，并且这些突变体中从突变位点释放 ADP 或另一个催化步骤受到损害。此外，我们的结果支持一个模型，其中 P-gp 的两个 NBD 在功能上并不等效。
In the nucleotide-binding domains (NBDs) of ABC transporters, such as mouse Mdr3 P-glycoprotein (P-gp), an invariant carboxylate residue (E552 in NBD1; E1197 in NBD2) immediately follows the Walker B motif (hyd(4)DE/D). Removal of the negative charge in mutants E552Q and E1197Q abolishes drug-stimulated ATPase activity measured by P-i release. Surprisingly, drug-stimulated trapping of 8-azido-[alpha-P-32]ATP is still observed in the mutants in both the presence and absence of the transition-state analogue vanadate (V-i), and ADP can be recovered from the trapped enzymes. The E552Q and E1197Q mutants show characteristics similar to those of the wild-type (WT) enzyme with respect to 8-azido-[alpha-P-32]ATP binding and 8-azido-[alpha-P-32]nucleotide trapping, with the latter being both Mg2+ and temperature dependent. Importantly, drug-stimulated nucleotide trapping in E552Q is stimulated by V-i and resembles the WT enzyme, while it is almost completely V-i insensitive in E1197Q. Similar nucleotide trapping properties are observed when aluminum fluoride or beryllium fluoride is used as an alternate transition-state analogue. Partial proteolytic cleavage of photolabeled enzymes indicates that, in the absence of V-i, nucleotide trapping occurs exclusively at the mutant NBD, whereas in the presence of V-i, nucleotide trapping occurs at both NBDs. Together, these results suggest that there is single-site turnover occurring in the E552Q and E1197Q mutants and that ADP release from the mutant site, or another catalytic step, is impaired in these mutants. Furthermore, our results support a model in which the two NBDs of P-gp are not functionally equivalent.