Phosphorylation and mutations of Ser16 in human phenylalanine hydroxylase -: Kinetic and structural effects
Phosphorylation and mutations of Ser16 in human phenylalanine hydroxylase -: Kinetic and structural effects
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DOI：
10.1074/jbc.m112197200
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发表时间：
2002-10-25
影响因子：
4.8
通讯作者：
Martínez, A
中科院分区：
文献类型：
--
作者：
Miranda, FF;Teigen, K;Martínez, A
环腺苷酸依赖性蛋白激酶对苯丙氨酸羟化酶（PAH）Ser（16）的磷酸化是一种翻译后修饰，可增加其基础活性并促进其被底物L-Phe激活。到目前为止，还没有关于柔性N-末端尾（残基1-18）的结构信息，包括磷酸化位点。为了进一步了解磷酸化对催化效率和酶稳定性的影响的分子基础，使用重组大鼠酶的晶体结构进行分子建模。由此获得的N-末端尾部的最可能的构象和取向表明，Ser（16）的磷酸化诱导局部构象变化，这是磷酸基团和Arg（13）之间的静电相互作用以及活性位点缝隙结构入口处环中Glu（280）的排斥的结果。N-末端尾残基（Met（1）-Leu（15））在磷酸化作用下的模型化重定向与观察到的构象变化和底物对活性位点的可及性增加一致，如圆二色性光谱和全长磷酸化和非磷酸化人PAH的酶动力学数据所示。为了进一步验证该模型，我们制备并表征了用带负电荷的残基取代Ser（16）的突变体，并发现S16 E在很大程度上模拟了人PAH磷酸化的作用。磷酸化的酶和具有酸性侧链而不是Ser的突变体（16）都显示出对有限的胰蛋白酶蛋白水解的抗性增加，并且如圆二色光谱所示，α-螺旋结构的含量增加。与模型结构一致，Arg（13）至Ser（16）磷酸盐桥的形成和N-末端尾部的构象变化也解释了磷酸化酶对有限胰蛋白酶蛋白水解的较高稳定性。用突变体R13 A和E381 A获得的结果进一步支持了所提出的通过磷酸化激活酶的分子机制的模型。
Phosphorylation of phenylalanine hydroxylase (PAH) at Ser(16) by cyclic AMP-dependent protein kinase is a post-translational modification that increases its basal activity and facilitates its activation by the substrate L-Phe. So far there is no structural information on the flexible N-terminal tail (residues 1-18), including the phosphorylation site. To get further insight into the molecular basis for the effects of phosphorylation on the catalytic efficiency and enzyme stability, molecular modeling was performed using the crystal structure of the recombinant rat enzyme. The most probable conformation and orientation of the N-terminal tail thus obtained indicates that phosphorylation of Ser(16) induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Arg(13) as well as a repulsion by Glu(280) in the loop at the entrance of the active site crevice structure. The modeled reorientation of the N-terminal tail residues (Met(1)-Leu(15)) on phosphorylation is in agreement with the observed conformational change and increased accessibility of the substrate to the active site, as indicated by circular dichroism spectroscopy and the enzyme kinetic data for the full-length phosphorylated and nonphosphorylated human PAH. To further validate the model we have prepared and characterized mutants substituting Ser(16) with a negatively charged residue and found that S16E largely mimics the effects of phosphorylation of human PAH. Both the phosphorylated enzyme and the mutants with acidic side chains instead of Ser(16) revealed an increased resistance toward limited tryptic proteolysis and, as indicated by circular dichroism spectroscopy, an increased content of alpha-helical structure. In agreement with the modeled structure, the formation of an Arg(13) to Ser(16) phosphate salt bridge and the conformational change of the N-terminal tail also explain the higher stability toward limited tryptic proteolysis of the phosphorylated enzyme. The results obtained with the mutant R13A and E381A further support the model proposed for the molecular mechanism for the activation of the enzyme by phosphorylation.