Effects of substitution at serine 40 of tyrosine hydroxylase on catecholamine binding.
Effects of substitution at serine 40 of tyrosine hydroxylase on catecholamine binding.
复制标题
酪氨酸羟化酶丝氨酸 40 处的取代对儿茶酚胺结合的影响。
DOI：
10.1021/bi010546d
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Fitzpatrick,PF
中科院分区：
文献类型：
--
作者：
McCulloch,RI;Daubner,SC;Fitzpatrick,PF
酪氨酸羟化酶调节结构域中Ser 40的磷酸化通过增加抑制性儿茶酚胺的解离速率来激活酶[Ramsey，A. J.，和菲茨帕特里克，P. F.（1998）Biochemistry37，8980 - 8986]。为了探索这种效应的结构基础，并确定其他氨基酸在功能上替代丝氨酸和丝氨酸磷酸的能力，测定了用其他氨基酸替代Ser 40的效应。只有轻微的变化，在theVmax值和theKm值酪氨酸和四氢蝶呤后，看到Ser 40替换丙氨酸，缬氨酸，苏氨酸，天冬氨酸，或谷氨酸，在磷酸化对稳态动力学参数的轻微影响。多巴胺和二羟基苯丙氨酸的结合更显着的影响。S40 T酶对任一种儿茶酚胺的亲和力与野生型酶的亲和力非常相似，而S40 E酶与磷酸化酶相似。S40 D酶对多巴的亲和力与磷酸化酶相当，但对多巴胺的亲和力高于后者。在两种儿茶酚胺的情况下，S40 V和S40 A酶显示出中等水平的活化。结果表明，丝氨酸羟基有助于稳定的儿茶酚胺抑制酶。此外，S40 E酶将是有用的，在酪氨酸羟化酶的多重磷酸化的影响的进一步研究，而丙氨酸酶不提供一个准确的模拟的未磷酸化的酶。
Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate of dissociation of inhibitory catecholamines [Ramsey, A. J., and Fitzpatrick, P. F. (1998)Biochemistry37, 8980−8986]. To probe the structural basis for this effect and to ascertain the ability of other amino acids to functionally replace serine and serine phosphate, the effects of replacement of Ser40 with other amino acids were determined. Only minor changes in theVmaxvalue and theKmvalues for tyrosine and tetrahydropterin were seen upon replacement of Ser40 with alanine, valine, threonine, aspartate, or glutamate, in line with the minor effects of phosphorylation on steady-state kinetic parameters. More significant effects were seen on the binding of dopamine and dihydroxyphenylalanine. The affinity of the S40T enzyme for either catecholamine was very similar to that of the wild-type enzyme, while the S40E enzyme was similar to the phosphorylated enzyme. The S40D enzyme had an affinity for DOPA comparable to the phosphorylated enzyme but a higher affinity for dopamine than the latter. With both catecholamines, the S40V and S40A enzymes showed intermediate levels of activation. The results suggest that the serine hydroxyl contributes to the stabilization of the catecholamine-inhibited enzyme. In addition, the S40E enzyme will be useful in further studies of the effects of multiple phosphorylation on tyrosine hydroxylase, while the alanine enzyme does not provide an accurate mimic of the unphosphorylated enzyme.