Orthophosphate binding at the dimer interface of Corynebacterium callunae starch phosphorylase: mutational analysis of its role for activity and stability of the enzyme.
Orthophosphate binding at the dimer interface of Corynebacterium callunae starch phosphorylase: mutational analysis of its role for activity and stability of the enzyme.
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DOI：
10.1186/1471-2091-11-8
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发表时间：
2010-01-29
期刊：
影响因子：
--
通讯作者：
Nidetzky B
中科院分区：
文献类型：
--
作者：
Mueller M;Nidetzky B
正磷酸盐在变构结合部位的识别是哺乳动物糖原磷酸化酶活性调节的一个关键特征。ALA分别取代了分布在非调控细菌淀粉磷酸化酶(来自愈伤棒状杆菌)二聚体界面上的三个结合位点上与正磷酸盐配位的蛋白质残基，以询问它们未知的功能对该酶的活性和稳定性的影响。与野生型酶相比，这些突变既不影响吡哆醛5‘-磷酸辅因子的含量，也不影响磷酸化酶制剂中的比活性，但它们破坏(Thr28→Ala，Arg141→Ala)或降低(Lys31→Ala，Ser174→Ala)正磷酸盐(10 mM或100 mM)对45°C失活和咪唑加强的亚基解离的异常强大的保护作用。突变的磷酸化酶的稳定性的丧失似乎主要是由于对正磷酸盐结合的亲和力减弱。在结晶学上观察到磷酸化酶在二聚体界面上的“非共价磷酸化”，硫酸盐的结合对酶的活性没有变构影响。Callunae淀粉磷酸化酶亚单位-亚单位界面上的磷酸位点似乎协同作用，为活性酶的天然二聚体结构提供了额外的动力学稳定性。据我们所知，用于四元结构稳定的分子策略在二聚体蛋白质中是新的。它可以清楚地区别于正磷酸盐对蛋白质热稳定性的共溶质效应，这是由于配体与蛋白质表面残基的(相对较弱的)相互作用造成的。
Orthophosphate recognition at allosteric binding sites is a key feature for the regulation of enzyme activity in mammalian glycogen phosphorylases. Protein residues co-ordinating orthophosphate in three binding sites distributed across the dimer interface of a non-regulated bacterial starch phosphorylase (from Corynebacterium callunae) were individually replaced by Ala to interrogate their unknown function for activity and stability of this enzyme. While the mutations affected neither content of pyridoxal 5'-phosphate cofactor nor specific activity in phosphorylase preparations as isolated, they disrupted (Thr28→Ala, Arg141→Ala) or decreased (Lys31→Ala, Ser174→Ala) the unusually strong protective effect of orthophosphate (10 or 100 mM) against inactivation at 45°C and subunit dissociation enforced by imidazole, as compared to wild-type enzyme. Loss of stability in the mutated phosphorylases appeared to be largely due to weakened affinity for orthophosphate binding. Binding of sulphate mimicking the crystallographically observed "non-covalent phosphorylation" of the phosphorylase at the dimer interface did not have an allosteric effect on the enzyme activity. The phosphate sites at the subunit-subunit interface of C. callunae starch phosphorylase appear to be cooperatively functional in conferring extra kinetic stability to the native dimer structure of the active enzyme. The molecular strategy exploited for quaternary structure stabilization is to our knowledge novel among dimeric proteins. It can be distinguished clearly from the co-solute effect of orthophosphate on protein thermostability resulting from (relatively weak) interactions of the ligand with protein surface residues.
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影响因子：
2.7
作者：
Nidetzky, B;Griessler, R;Tanfani, F
通讯作者：
Tanfani, F
影响因子：
4.1
作者：
Schwarz, A;Pierfederici, FM;Nidetzky, B
通讯作者：
Nidetzky, B
影响因子：
2.9
作者：
SHALTIEL, S;HEDRICK, JL;FISCHER, EH
通讯作者：
FISCHER, EH
影响因子：
2.9
作者：
DRUECKES, P;SCHINZEL, R;PALM, D
通讯作者：
PALM, D
影响因子：
8
作者：
Griessler, R;D'Auria, S;Nidetzky, B
通讯作者：
Nidetzky, B