Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase
Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase
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DOI：
10.1110/ps.4560102
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发表时间：
2002-08-01
期刊：
影响因子：
8
通讯作者：
Kobe, B
中科院分区：
文献类型：
--
作者：
Horne, J;Jennings, IG;Kobe, B
苯丙氨酸羟基酶(PAH)由底物苯丙氨酸激活，并通过N端自身调节序列中16位cAMP依赖的蛋白激酶的磷酸化而被激活。磷酸化和非磷酸化形式的酶的晶体结构表明，在没有苯丙氨酸的情况下，两种情况下包括磷酸化位置的N-末端18个残基都不包含可解释的电子密度。我们使用核磁共振(核磁共振)光谱来表征分子的这个N-末端区域在调节途径的不同阶段。在具有完整N-末端区域的PAH中观察到了许多尖锐的共振，但在缺少N-末端29个残基的截断突变体中没有观察到尖锐的共振。因此，N-末端序列代表分子的可移动柔性区域。在加入苯丙氨酸后，共振变得更弱，表明失去了迁移率。对应于多环芳烃第2-20位残基的多肽在磷酸化和非磷酸化形式下具有不同的结构特征，前者表现出增加的二级结构。我们的结果支持这样的模型，即在苯丙氨酸结合时，PAH的移动N-末端18个残基与分子的折叠核心结合；磷酸化可能促进这种相互作用。
Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser 16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.