Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation.
Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation.
复制标题
通过甘氨酸 185 向缬氨酸突变改善人 P-糖蛋白的能量耦合。
DOI：
10.1021/bi035365l
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发表时间：
2004
期刊：
影响因子：
2.9
通讯作者：
Al-Shawi,MarwanK
中科院分区：
文献类型：
--
作者：
Omote,Hiroshi;Figler,RobertA;Polar,MarkK;Al-Shawi,MarwanK
人P-糖蛋白（ABCB 1，MDR 1）的甘氨酸185到缬氨酸突变先前已从高秋水仙素抗性细胞系中分离出来。我们已经使用了在酿酒酵母中表达的纯化和重建的P-糖蛋白[Figler et al.（2000）Arch.Biochem.Bioprotein.376，34 - 46]，并设计了一组热力学分析来揭示提高抗性的机制。纯化的G185 V酶显示改变的基础ATP酶活性，但秋水仙碱和依托泊苷依赖性活动的强烈刺激，这表明这些药物对ATP酶活性的严格调节。突变体对秋水仙碱的Km值高于野生型，对依托泊苷的Km值低于野生型。其他运输药物的动力学常数没有显着修改这种突变。系统的热力学分析表明，G185 V酶具有修改的热力学性质的秋水仙碱和依托泊苷依赖的活动。为了提高秋水仙碱或依托泊苷转运的速率，G185 V酶降低了转运限速步骤的Arrhenius活化能。当转运依托泊苷或秋水仙碱时，野生型P-糖蛋白的高过渡态能量增加了在没有转运的情况下过渡态非生产性降解的可能性。G185 V P-糖蛋白转运依托泊苷或秋水仙碱在一个积极的更有效的方式与减少的活化能的熵和熵分量。我们的新数据完全调和了以前研究中明显矛盾的结果。自旋标记的G185 C酶在半胱氨酸的背景和动力学参数的G185 C酶的EPR分析表明，位置185被其他残基包围，是体积敏感的。这些结果和原子细节结构建模表明，残基185是一个关键点，在传输催化位点和秋水仙碱药物结合域之间的构象变化。用大体积的缬氨酸替换该残基改变了这种通信，并导致依托泊苷或秋水仙碱更有效的运输。
A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed inSaccharomyces cerevisiae[Figler et al. (2000)Arch. Biochem. Biophys.376, 34−46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparentKmfor colchicine and a lowerKmfor etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this residue with a bulky valine alters this communication and results in more efficient transport of etoposide or colchicine.