Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.
Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.
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DOI：
10.1021/bi501183x
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发表时间：
2014-12-16
期刊：
影响因子：
2.9
通讯作者：
Fitzpatrick, Paul F.
中科院分区：
文献类型：
--
作者：
Roberts, Kenneth M.;Khan, Crystal A.;Hinck, Cynthia S.;Fitzpatrick, Paul F.
苯丙氨酸羟化酶（PheH）是一种肝酶，催化饮食中过量的苯丙氨酸羟基化为酪氨酸，被苯丙氨酸激活。在低水平的苯丙氨酸下缺乏活性归因于蛋白质的调节结构域的N-末端通过阻断底物接近活性位点而充当抑制肽。苯丙氨酸结合激活酶的位点的位置是未知的，并且已经提出了催化结构域中的活性位点和N-末端调节结构域中的单独位点。结合的活性位点铁的儿茶酚胺被用来探测的活性位点的可访问性。去除的监管结构域增加的速率常数协会的几种儿茶酚胺与野生型酶的0.02倍。通过将活性位点残基Arg 270突变为赖氨酸，有效地消除了活性位点中苯丙氨酸的结合。突变酶的kcat/Kphe值下降104，突变酶对苯丙氨酸的Km值>0.5 M。R270 K酶与苯丙氨酸的孵育也导致儿茶酚胺结合的速率常数增加2倍。在野生型酶中观察到的色氨酸荧光发射光谱在被苯丙氨酸激活后的变化也在存在苯丙氨酸的R270 K突变酶中观察到。这两个结果都证实了苯丙氨酸对PheH的活化不需要结合活性位点中的氨基酸。这与可能在调节域中的单独的变构位点一致。
Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.
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