FURTHER-STUDIES OF THE ROLE OF SER-16 IN THE REGULATION OF THE ACTIVITY OF PHENYLALANINE-HYDROXYLASE
FURTHER-STUDIES OF THE ROLE OF SER-16 IN THE REGULATION OF THE ACTIVITY OF PHENYLALANINE-HYDROXYLASE
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DOI：
10.1073/pnas.92.11.4743
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发表时间：
1995-05-23
影响因子：
11.1
通讯作者：
KAUFMAN, S
中科院分区：
文献类型：
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作者：
KOWLESSUR, D;YANG, XJ;KAUFMAN, S
先前提出，通过cAMP依赖性蛋白激酶介导的Ser-16的磷酸化激活大鼠肝苯丙氨酸羟化酶（EC 1.14.16.1）是由于引入带负电荷的磷酸基团。为了探索该提议的有效性，我们应用定点诱变，用带负电荷的氨基酸谷氨酸和天冬氨酸特异性地取代Ser-16;用极性不带电荷的氨基酸天冬酰胺和谷氨酰胺特异性地取代Ser-16;用带正电荷的氨基酸赖氨酸特异性地取代Ser-16。将野生型和突变体酶纯化至同质，并通过比较野生型羟化酶的磷酸化形式与突变体的磷酸化形式的活化状态来检查Ser-16在苯丙氨酸羟化酶活化中的重要性，对野生型磷酸化羟化酶进行的动力学研究表明，所有的活化都可以通过苯丙氨酸或蝶呤辅因子的V-max的增加来解释，而K-m没有变化，用带负电荷的残基谷氨酸或天冬氨酸取代Ser-16，导致羟化酶活化2- 4倍，而用谷氨酰胺、天冬酰胺、赖氨酸或丙氨酸取代则导致更温和的增加。此外，发现溶血素刺激磷酸化羟化酶和突变体酶S16 E和S16 D 6-7倍。相反，突变体S16 Q、S16 N和S16 A都显示出与野生型相同的激活幅度。这项研究表明，cAMP依赖性蛋白激酶对Ser-16的磷酸化激活该酶是由于负电荷的引入并强烈表明羟化酶的调节和催化结构域之间的静电相互作用的参与。
It was previously proposed that the activation of rat liver phenylalanine hydroxylase (EC 1.14.16.1) by cAMP-dependent protein kinase-mediated phosphorylation of Ser-16 is due to the introduction of the negatively charged phosphate group, To explore the validity of this proposal, we have applied site-directed mutagenesis to specifically replace Ser-16 with negatively charged amino acids, glutamic and aspartic; with polar uncharged amino acids, asparagine and glutamine; with the positively charged amino acid lysine; and with the nonpolar hydrophobic amino acid alanine, The wild-type and mutant enzymes were purified to homogeneity, and the importance of Ser-16 in the activation of phenylalanine hydroxylase was examined by comparing the state of activation of the phosphorylated form of the wild-type hydroxylase with that of the mutants, The kinetic studies carried out on the wild-type phosphorylated hydroxylase showed that all the activation could be accounted for by an increase in V-max with no change in K-m for either phenylalanine or the pterin cofactor, Replacement of Ser-16 with a negatively charged residue, glutamate of aspartate, resulted in the activation of the hydroxylase by 2- to 4-fold, whereas replacement with glutamine, asparagine, lysine, or alanine resulted in a much more modest increase. Further, lysolecithin was found to stimulate the phosphorylated hydroxylase and the mutant enzymes S16E and S16D by a factor of 6-7, In contrast, the mutants S16Q, S16N, and S16A all showed the same magnitude of activation as the wild-type with lysolecithin, Therefore, this study demonstrates that activation of the enzyme by phosphorylation of Ser-16 by cAMP-dependent protein kinase is due to the introduction of negative charge(s) and strongly suggests the involvement of electrostatic interaction between the regulatory and catalytic domains of the hydroxylase.