Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate.
Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate.
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DOI：
10.1016/s0021-9258(19)38459-5
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发表时间：
1990-07
期刊：
影响因子：
--
通讯作者：
K. Ahn;A. Kornberg
中科院分区：
文献类型：
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作者：
K. Ahn;A. Kornberg
多磷酸激酶（PPK）在自由可逆反应中将ATP的末端磷酸盐聚合成长链多磷酸盐（聚（P）或（Pi）n）（Kornberg，S. R.（1957）Biochim. Biophys. Acta 26，294-300），平衡nADP +（Pi）n，PPK中的nATP，现在纯化至均一，是69-kDa亚基的四聚体。在合成反应中不需要添加引物，ATP或无机正磷酸盐（Pi）也不起此作用。PPK在聚（P）合成的条件下自磷酸化; Pi通过其酸不稳定性和碱稳定性判断通过氮-磷酸键连接。在合成反应中加入ATP时，磷酸盐从分离的磷酸酶掺入聚（P）中，加入ADP时，磷酸盐掺入ATP中，表明磷酸酶是反应中的中间体。在5 μ M的ATP水平下，远低于其2 mM的Km，聚（P）合成的显著滞后可以通过四聚磷酸盐而不是Pi、PPi或三聚磷酸盐去除。这种刺激作用的基础尚不清楚，因为四聚磷酸盐不促进假定的磷酸酶中间体的去磷酸化。
Polyphosphate kinase (PPK) polymerizes the terminal phosphate of ATP to a long chain polyphosphate (poly(P) or (Pi)n) in a freely reversible reaction (Kornberg, S. R. (1957) Biochim. Biophys. Acta 26, 294-300), nATP in equilibrium nADP + (Pi)n, PPK, now purified to homogeneity, is a tetramer of 69-kDa subunits. Addition of a primer in the synthetic reaction is not required, nor does ATP or inorganic orthophosphate (Pi) serve in this role. PPK is autophosphorylated under the conditions of poly(P) synthesis; Pi is linked by a nitrogen-phosphate bond as judged by its acid lability and alkali stability. Incorporation of phosphate from the isolated phosphoenzyme into poly(P) upon the addition of ATP in the synthetic reaction and its incorporation into ATP upon the addition of ADP indicate phosphoenzyme to be an intermediate in the reaction. At an ATP level of 5 microM, well below its Km of 2 mM, a pronounced lag in poly(P) synthesis can be removed by tetrapolyphosphate but not by Pi, PPi, or tripolyphosphate. The basis for this stimulatory effect is not clear inasmuch as tetrapolyphosphate does not promote the dephosphorylation of the presumed phosphoenzyme intermediate.