Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
BMC Biochem. 2010 Jan 29;11:8. doi: 10.1186/1471-2091-11-8.
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 degrees 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.
在变构结合位点识别正磷酸盐是调节哺乳动物糖原磷酸化酶酶活性的关键特征。在非调节细菌淀粉磷酸化酶（来自棒状杆菌）的二聚体界面上分布的三个结合位点中，与正磷酸盐配位的蛋白质残基分别被丙氨酸取代，以研究其对该酶活性和稳定性的未知功能。
虽然突变既不影响吡哆醛 5'-磷酸辅因子的含量，也不影响作为分离物的磷酸化酶制剂的比活性，但它们破坏了（Thr28-->Ala，Arg141-->Ala）或降低了（Lys31-->Ala，Ser174-->Ala）正磷酸盐（10 或 100 mM）对 45°C 失活和咪唑强制亚基解离的异常强保护作用，与野生型酶相比。突变磷酸化酶的稳定性丧失似乎主要是由于与正磷酸盐结合的亲和力减弱所致。硫酸盐的结合模拟了在二聚体界面处观察到的磷酸化酶的“非共价磷酸化”，对酶活性没有变构效应。
C. callunae 淀粉磷酸化酶亚基-亚基界面上的磷酸盐位点似乎在赋予天然二聚体结构的额外动力学稳定性方面具有协同功能。用于四级结构稳定的分子策略在我们的知识中是新颖的，它可以与配体与蛋白质表面残基的（相对较弱）相互作用导致的正磷酸盐对蛋白质热稳定性的共溶质效应明显区分开来。
