Pyrococcus abyssi alkaline phosphatase: the dimer is the active form.
Pyrococcus abyssi alkaline phosphatase: the dimer is the active form.
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深渊火球菌碱性磷酸酶：二聚体是活性形式。
DOI：
10.1016/j.jinorgbio.2004.01.009
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Kantrowitz,ER
中科院分区：
文献类型：
--
作者：
Zappa,S;Boudrant,J;Kantrowitz,ER
碱性磷酸酶(APs)，E.C.3.1.3.1，是一种在碱性条件下最具活性的非特异性磷酸单酯酶。它们经典地被认为是同源二聚体金属酶。这种四元结构一直被认为是活性所必需的，尽管四元结构和活性之间的关系还没有被很好地理解。重组深海热球菌AP已被分离和鉴定，在天然聚丙烯酰胺凝胶电泳图上显示具有两个活性的四元结构：一个单体和一个同源二聚体。本工作的目的是通过分离两种四元形式并建立控制二聚体组装和解离的参数来确定深海假单胞菌AP在溶液中的实际四元结构。PH似乎是一个重要的参数：在酸性介质中，单体/二聚体的比例向单体移动。缓冲液组成也影响四元结构：在相同pH条件下，在磷酸二氢钾缓冲液中观察到两种四元结构，而在三(羟甲基)氨基甲烷盐酸盐缓冲液中只观察到二聚体。发现与该酶结合的金属参与了该四元结构的稳定。事实上，去除金属后得到的深海假单胞菌AP是单体。后者的重新激活是以可变的效率实现的。从这些实验中，没有分离到任何活性单体，从而得出结论：深海假单胞菌AP的活性形式是同源二聚体。
Alkaline phosphatases (APs), E.C. 3.1.3.1, are non-specific phosphomonoesterases optimally active under alkaline conditions. They are classically known to be homodimeric metalloenzymes. This quaternary structure has been considered necessary for activity, although the relationship between quaternary structure and activity is not well understood. Recombinant Pyrococcus abyssi AP was previously isolated and characterized, appearing to have two active quaternary structures on native polyacrylamide gel electrophoresis: a monomer and a homodimer. The purpose of the present work was to determine the actual quaternary structure of P. abyssi AP in solution, by isolating each of the two quaternary forms and establishing the parameters governing the assembly and dissociation of the dimer. pH appeared to be an important parameter: in acidic media, the monomer/dimer ratio shifted towards monomer. Buffer composition also affected the quaternary structure: at the same pH, in potassium phosphate buffer, the two quaternary structures were observed, whereas in tris(hydroxymethyl)aminomethane hydrochloride buffer, only the dimer was observed. Metals bound to the enzyme were found to be involved in the stability of the quaternary structure. Indeed, the P. abyssi AP obtained upon removal of the metals was monomeric. Reactivation of the latter was achieved with variable efficiency. From these experiments, no active monomer could be isolated, leading the conclusion that the active form of P. abyssi AP is the homodimer.
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DOI：
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发表时间：
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期刊：
影响因子：
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DOI：
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DOI：
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