Regulation of tryptophan synthase by temperature, monovalent cations, and an allosteric ligand. Evidence from arrhenius plots, absorption spectra, and primary kinetic isotope effects
Regulation of tryptophan synthase by temperature, monovalent cations, and an allosteric ligand. Evidence from arrhenius plots, absorption spectra, and primary kinetic isotope effects
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DOI：
10.1021/bi9921586
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发表时间：
2000-04-25
期刊：
影响因子：
2.9
通讯作者：
Miles, EW
中科院分区：
文献类型：
--
作者：
Fan, YX;McPhie, P;Miles, EW
为了研究酶构象和催化之间的联系，我们确定了在不存在或存在不同单价阳离子（Cs+、Na+和CuH+）以及变构配体α-甘油3-磷酸的情况下，温度对色氨酸合酶α(2)β(2)复合物和β(2)亚基催化特性的影响。在某些配体存在的情况下，5 到 50 摄氏度之间的活性数据的阿伦尼乌斯图是非线性的，但在其他配体存在的情况下则不然。产生非线性阿伦尼乌斯图的条件也会在酶-底物中间体的平衡分布和初级动力学同位素效应中产生温度依赖性变化。结果证明非线性阿累尼乌斯图是由催化限速步骤之前的温度依赖性构象变化引起的。与构象变化相关的数据的热力学分析表明，低温下的活化能比高温下高得多。我们将结果与模型相关联，在该模型中，酶在某些条件下通过升高温度从低活性“开放”构象转变为高活性“闭合”构象。变构配体和不同的单价阳离子，包括也充当离液剂的 GuH(+)，影响开放和封闭形式之间的平衡。构象转换中的大的正熵变化表明，封闭构象是由加强的疏水相互作用导致的，该相互作用将水排除在β亚基的活性位点之外。
To investigate the linkage between enzyme conformation and catalysis, we have determined the effects of temperature on catalytic properties of the tryptophan synthase alpha(2)beta(2) complex and beta(2) subunit in the absence or presence of different monovalent cations (Cs+, Na+, and CuH+) and of an allosteric ligand, alpha-glycerol 3-phosphate. Arrhenius plots of the activity data between 5 and 50 degrees C are nonlinear in the presence of certain ligands but not others. The conditions that yield nonlinear Arrhenius plots also yield temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. The results provide evidence that the nonlinear Arrhenius plots are caused by a temperature-dependent conformational change that precedes the rate-limiting step in catalysis. Thermodynamic analysis of the data associated with the conformational change shows that the activation energies are much higher at low temperatures than at high temperatures. We correlate the results with a model in which the enzyme is converted by increased temperature under certain conditions from a low-activity "open" conformation to a high-activity "closed" conformation. The allosteric Ligand and different monovalent cations, including GuH(+), which also acts as a chaotropic agent, affect the equilibrium between the open and closed forms. The large positive entropy changes in the conformational conversion suggest that the closed conformation results from tightened hydrophobic interactions that exclude water from the active site of the beta subunit.