Mahaney J E, Froehlich J P, Thomas D D
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455, USA.
Biochemistry. 1995 Apr 11;34(14):4864-79. doi: 10.1021/bi00014a044.
We have used time-resolved electron paramagnetic resonance (EPR) and quenched-flow kinetics in order to investigate the dynamics of Ca-ATPase conformational changes involved in Ca2+ pumping in sarcoplasmic reticulum (SR) membranes at 2 degrees C. The Ca-ATPase was selectively labeled with an iodoacetamide spin label (IASL), which yields EPR spectra sensitive to enzyme conformational changes during ATP induced enzymatic cycling. The addition of ATP, AMPPCP, CrATP, or ADP decreased the rotational mobility of a fraction of the probes, indicating a distinct protein conformational state corresponding to this probe population, while Pi under conditions producing "backdoor" phosphorylation produced no spectral change. Transient changes in the amplitude of the restricted component associated with the pre-steady state of Ca2+ pumping were detected with 10 ms time resolution after an [ATP] jump produced by laser flash photolysis of caged ATP in the EPR sample. The laser energy was adjusted to generate 100 microM ATP from 1 mM caged ATP. At 0.1 M KCl, the EPR transient consisted of a brief initial lag phase, a monoexponential phase with a rate of 20 s-1, and a decay back to the initial intensity after the ATP had been consumed. Raising [KCl] from 0.1 to 0.4 M slowed the rate of the exponential phase from 20 to 6 s-1. Lowering the pH from 7 to 6, which increased the rate of caged ATP photolysis, eliminated the lag but did not change the apparent rate of the EPR signal rise. Parallel acid quenched-flow experiments conducted at 0.1 M KCl and 100 microM ATP produced fast (50-58 s-1) and slow (20 s-1) phases of phosphoenzyme formation. Increasing [KCl] from 0.1 to 0.4 M decreased the rate of the slow phase of phosphorylation from 20 to 5 s-1, without affecting the fast phase. The close correlation between the slow phase of phosphorylation and the exponential phase of the EPR signal suggests that the spin probe monitors a conformational event associated with phosphoenzyme formation in a population of catalytic sites with delayed kinetics. We propose that this constraint is imposed by conformational coupling between the catalytic subunits in a Ca-ATPase oligomer and that, consequently, the EPR signal reflects changes in quaternary protein structure as well as changes in secondary and tertiary structure associated with ATP-dependent phosphorylation.
我们运用时间分辨电子顺磁共振（EPR）和猝灭流动力学，以研究2℃下肌浆网（SR）膜中参与Ca2+泵浦的Ca - ATP酶构象变化的动力学。Ca - ATP酶用碘乙酰胺自旋标记（IASL）进行选择性标记，该标记产生的EPR谱对ATP诱导的酶促循环过程中的酶构象变化敏感。添加ATP、AMPPCP（腺苷5'-（β，γ-亚甲基）三磷酸）、CrATP（铬（III）-腺苷5'-三磷酸）或ADP会降低一部分探针的旋转流动性，表明存在一种与该探针群体相对应的独特蛋白质构象状态，而在产生“后门”磷酸化的条件下添加Pi则不会引起光谱变化。在EPR样品中通过激光闪光光解笼形ATP产生[ATP]跃变后，以10毫秒的时间分辨率检测到与Ca2+泵浦预稳态相关的受限成分振幅的瞬态变化。调节激光能量以从1 mM笼形ATP产生100 μM ATP。在0.1 M KCl条件下，EPR瞬态由一个短暂的初始滞后阶段、一个速率为20 s-1的单指数阶段以及ATP消耗后衰减回到初始强度组成。将[KCl]从0.1 M提高到0.4 M会使指数阶段的速率从20 s-1减慢至6 s-1。将pH从7降低到6，这增加了笼形ATP的光解速率，消除了滞后现象，但并未改变EPR信号上升的表观速率。在0.1 M KCl和100 μM ATP条件下进行的平行酸猝灭流实验产生了快速（50 - 58 s-1）和慢速（20 s-1）的磷酸酶形成阶段。将[KCl]从0.1 M增加到0.4 M会使磷酸化的慢速阶段速率从20 s-1降低至5 s-1，而不影响快速阶段。磷酸化的慢速阶段与EPR信号的指数阶段之间的密切相关性表明，自旋探针监测的是一群具有延迟动力学的催化位点中与磷酸酶形成相关的构象事件。我们提出这种限制是由Ca - ATP酶寡聚体中催化亚基之间的构象偶联施加的，因此，EPR信号反映了四级蛋白质结构的变化以及与ATP依赖性磷酸化相关的二级和三级结构的变化。
