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首页> 外文期刊>Biochemistry >Detection of conformational changes along the kinetic pathway of protein kinase A using a catalytic trapping technique.
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Detection of conformational changes along the kinetic pathway of protein kinase A using a catalytic trapping technique.

机译：使用催化捕获技术检测沿蛋白激酶A动力学路径的构象变化。

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摘要

The dissociation rate constants for the two products of the reaction catalyzed by protein kinase A, ADP and phosphopeptide, were measured using a catalytic trapping technique to determine the role of product release in enzyme turnover. The enzyme was preequilibrated with ADP, and the reaction was initiated with a peptide substrate, LRRASLG, and ATP in a rapid quench flow instrument. At high, free magnesium concentrations (>2 mM), the large 'burst' in phosphopeptide production disappears, and, at low concentrations of free magnesium (0.5-1 mM), the kinetic transients become sigmoidal prior to the linear turnover phase. Increasing the concentrations of ATP or ADP did not influence the shape of the kinetic transients in the first 20 ms. ADP preequilibration protects the enzyme from inhibition by the covalent inactivator p-fluorosulfonylbenzoyl 5'-adenosine at 0.5 mM free magnesium, indicating that a competent E. ADP complex forms at low metal concentrations and the sigmoidal behavior in the catalytic trapping experiment is not due to free enzyme at high ATP concentrations. Simulations of the data indicate that ADP release is rate-limiting for turnover at high magnesium concentrations, but, at lower physiological levels of 0.5 and 1 mM, the off rate of ADP is 3- and 2-fold higher than k(cat), respectively. In contrast, the initial portions of the kinetic transients at 0.5 mM free magnesium were unaffected by phosphopeptide preequilibration, indicating that the release rate of this product is significantly larger than turnover. The transient kinetic data, coupled with a previous report [Shaffer and Adams (1999) Biochemistry 38, 5572-5581], support a phosphorylation mechanism under physiological magnesium concentrations that incorporates two partially rate-determining conformational changes, one prior to and one after the phosphoryl transfer step. We propose that the initial step activates the enzyme through key positioning of one or more active-site residues and the second step relaxes this conformation, a prerequisite for a subsequent catalytic cycle.
机译：使用催化捕集技术测定蛋白激酶A，ADP和磷酸肽催化的两种反应产物的解离速率常数，以确定产物释放在酶转化中的作用。酶用ADP预先平衡，并在快速淬灭流动仪中以肽底物，LRRASLG和ATP启动反应。在高游离镁浓度（> 2 mM）下，磷酸肽生产中的大“爆发”消失，而在低浓度游离镁（0.5-1 mM）下，动力学瞬变在线性转换阶段之前变为S形。增加ATP或ADP的浓度不会影响前20 ms的动力学瞬变形状。 ADP预平衡可保护酶免于在0.5 mM游离镁下被共价灭活剂对氟磺酰基苯甲酰基5'-腺苷抑制，这表明在低金属浓度下可形成有效的E. ADP复合物，而在催化捕集实验中的S形行为并非由于ATP浓度高时的游离酶。数据模拟表明，在高镁浓度下，ADP释放是限速的，但在较低的生理水平（0.5和1 mM）下，ADP的释放速率比k（cat）高3到2倍，分别。相反，在0.5 mM游离镁处的动力学瞬变的初始部分不受磷酸肽预平衡的影响，表明该产物的释放速率明显大于周转率。瞬态动力学数据与以前的报道[Shaffer and Adams（1999）Biochemistry 38，5572-5581]结合在一起，支持了在生理镁浓度下的磷酸化机制，该机制结合了两个部分决定速率的构象变化，一个在改变前，一个在改变后。磷酰基转移步骤。我们建议，第一步通过一个或多个活性位点残基的关键定位来激活酶，而第二步则放松这种构象，这是后续催化循环的前提。

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