Investigations of ligand association and dissociation rates in the "open" and "closed" states of myoglobin.
Investigations of ligand association and dissociation rates in the "open" and "closed" states of myoglobin.
复制标题
研究肌红蛋白“开放”和“闭合”状态下的配体缔合和解离速率。
DOI：
10.1006/jmbi.1993.1491
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发表时间：
1993
影响因子：
5.6
通讯作者：
Champion,PM
中科院分区：
文献类型：
--
作者：
Tian,WD;Sage,JT;Champion,PM
动力学和拉曼光谱研究相结合，分析配体结合和解离率作为pH值在肌红蛋白水溶液的功能。双脉冲闪光光解方案用于动态选择肌红蛋白集合的快速再结合（开放口袋）部分，并确定“开放”和“封闭”远端口袋蛋白结构之间的平均时间尺度（~ 1 ~ 10 μs）。由于这个时间尺度比配体从溶液迁移到血红素口袋的速度快（~ 10-4s），因此可以使用时间平均种群分析，而不是状态的叠加，来描述配体的结合和解离动力学。拉曼光谱提供了作为pH值函数的开放和关闭远端口袋态的相对种群，与动力学测量并行，用于确定配体结合和解离特定于这些状态的速率。在293 K （1 mM CO）的水溶液中，k0on= 5.6 × 102s-1, k0off= 8.5 × 10-2s-1, k1on= 5.0 × 102s-1,k1off= 1.3 × 10-2s-1。开放形式的解离和缔合率的数量级增加表明，它可能在配体结合过程中发挥重要作用，即使它在pH 7下仅占时间平均种群的~ 5%。对于293 K （1.36 mM O2）下的氧结合，我们发现k0on= 4.6 × 104s-1,k0off ~ 104±2s-1, k1on= 2.0 × 104s-1,k1off= 13 s-1。开放形式解离速率的急剧增加可能是由于失去了与远端组氨酸的氢键，该氢键使结合的o2稳定在封闭状态。总的来说，这些结果表明，开放构象在决定配体结合和解离速率方面起着重要作用，并表明环境诱导的开放群体调节可以作为肌肉细胞摄取和输送氧气的生物分子控制机制。
Kinetic and Raman spectroscopic studies are combined to analyze ligand association and dissociation rates as a function of pH in aqueous solutions of myoglobin. A double-pulse flash photolysis protocol is used to kinetically select a rapidly rebinding (open pocket) fraction of the myoglobin ensemble and determine the timescale for averaging (∼ 1 to 10 μs) between the "open" and "closed" distal pocket protein conformations. Since this timescale is fast compared to the rate of ligand migration from the solution to the heme pocket (∼ 10-4s), a time-averaged population analysis, rather than a superposition of states, can be used to describe the ligand association and dissociation kinetics. Raman spectroscopy provides the relative populations of the open and closed distal pocket states as a function of pH which, in parallel with kinetics measurements, are used to determine the rates for ligand association and dissociation specific to these states. In aqueous solution at 293 K (1 mM CO) we findk0on= 5·6 × 103s-1,k0off= 8·5 × 10-2s-1for the open state andk1on= 5·0 × 102s-1,k1off= 1·3 × 10-2s-1for the closed state. The order of magnitude increase in the dissociation and association rates of the open form suggests that it may play a significant role in the ligand binding process, even though it comprises only ∼ 5% of the time-averaged population at pH 7. For oxygen binding at 293 K (1·36 mM O2) we findk0on= 4·6 × 104s-1,k0off∼ 104±2s-1for the open state andk1on= 2·0 × 104s-1,k1off= 13 s-1for the closed state. The dramatic increase in the dissociation rate of the open form is probably due to the loss of the hydrogen bond with the distal histidine, which stabilizes the bound O2in the closed state. Overall, these results demonstrate that the open conformation plays a significant role in determining the ligand association and dissociation rates and suggest that environmentally induced modulations of the open population could be used as a biomolecular control mechanism for the uptake and delivery of oxygen in muscle cells.