Khan Crystal A, Fitzpatrick Paul F
Department of Biochemistry and Structural Biology , University of Texas Health Science Center , San Antonio , Texas 78229 , United States.
Biochemistry. 2018 Nov 6;57(44):6274-6277. doi: 10.1021/acs.biochem.8b00919. Epub 2018 Oct 24.
Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that is activated by phenylalanine. The enzyme is also phosphorylated by protein kinase A, but the effects of phosphorylation are unclear. Recent structural studies ( Meisburger et al. ( 2016 ) J. Amer. Chem. Soc. 138 , 6506 - 6516 ) support a model in which activation of the enzyme involves dimerization of the regulatory domains, creating the allosteric site for phenylalanine at the dimer interface. This conformational change also results in a change in the fluorescence of the protein that can be used to monitor activation. The kinetics of activation of PheH are biphasic over a range of phenylalanine concentrations. These data are well-described by a model involving an initial equilibrium between the resting form and the activated conformation, with a value of the equilibrium constant for formation of the activated conformation, L, equal to 0.007, followed by binding of two molecules of phenylalanine. Phosphorylation increases L 10-fold by increasing the rate constant for conversion of the resting form to the activated form. The results provide functional support for the previous structural model, identify the specific effect of phosphorylation on the enzyme, and rationalize the lack of change in the protein structure upon phosphorylation.
肝脏苯丙氨酸羟化酶（PheH）是一种变构酶，可被苯丙氨酸激活。该酶也会被蛋白激酶A磷酸化，但其磷酸化的作用尚不清楚。最近的结构研究（Meisburger等人，（2016）《美国化学会志》138，6506 - 6516）支持一种模型，即该酶的激活涉及调节结构域的二聚化，在二聚体界面处形成苯丙氨酸的变构位点。这种构象变化也会导致蛋白质荧光的改变，可用于监测激活过程。在一系列苯丙氨酸浓度范围内，PheH激活的动力学是双相的。这些数据通过一个模型得到了很好的描述，该模型涉及静止形式和激活构象之间的初始平衡，激活构象形成的平衡常数L的值等于0.007，随后是两个苯丙氨酸分子的结合。磷酸化通过增加静止形式转化为激活形式的速率常数，使L增加了10倍。这些结果为之前的结构模型提供了功能支持，确定了磷酸化对该酶的具体作用，并解释了磷酸化后蛋白质结构缺乏变化的原因。
