Phosphoprotein with phosphoglycerate mutase activity from the archaeon Sulfolobus solfataricus
Phosphoprotein with phosphoglycerate mutase activity from the archaeon Sulfolobus solfataricus
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DOI：
10.1128/jb.185.7.2112-2121.2003
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发表时间：
2003-04-01
影响因子：
3.2
通讯作者：
Kennelly, PJ
中科院分区：
文献类型：
--
作者：
Ben Potters, M;Solow, BT;Kennelly, PJ
当极端酸性嗜热古细菌 Sulfiblobus solfataricus 的可溶性提取物与 [gamma-P-32]ATP 一起孵育时，几种蛋白质被放射性标记。其中比较突出的一种，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 上以类似于 46 kDa 的质量迁移，通过柱色谱和 SDS-PAGE 进行纯化，并通过 Edman 技术和质谱进行氨基酸序列分析。与所获得的部分序列的最佳匹配是开放阅读框 sso0417 的潜在多肽产物，其 DNA 衍生的氨基酸序列显示出许多特征，让人想起 2,3-二磷酸甘油酸独立的磷酸甘油酸 (PGA) 变位酶 [iPGM]。因此克隆了开放阅读框sso0417，并在大肠杆菌中表达了其蛋白产物。对其催化能力的测定表明，该蛋白质是一种中等有效的 PGA 变位酶，也表现出低水平的磷酸水解酶活性。 PGA 变位酶活性取决于二价金属离子（例如 Co2+ 或 Mn2+）的存在。当与[γ-P-32]ATP 或[γ-P-32]GTP 一起孵育时，重组蛋白发生自磷酸化。磷酸化位点被确定为 Ser(59)，它对应于细菌和真核 iPGM 中催化必需的丝氨酸残基。磷酸酶中间体的化学和动力学表现良好。 P-32 标记的磷酸酶与 3-PGA 一起孵育导致放射性磷酸盐消失，同时 P-32 标记的 PGA 出现，其速率与 PGA 变位酶活性稳态测定中测量的速率相当。
When soluble extracts of the extreme acidothermophilic archaeon Sulfiblobus solfataricus were incubated with [gamma-P-32]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of similar to46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co2+ or Mn2+. The recombinant protein underwent autophosphorylation when incubated with either [gamma-P-32]ATP or [gamma-P-32]GTP. The site of phosphorylation was identified as Ser(59), which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs. The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner. Incubation of the P-32-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of P-32-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.