Plateau P, Fromant M, Schmitter J M, Blanquet S
Laboratoire de Biochimie, Unité de Recherche Associée 240 Centre National de la Recherche Scientifique, Ecole Polytechnique, Palaiseau, France.
J Bacteriol. 1990 Dec;172(12):6892-9. doi: 10.1128/jb.172.12.6892-6899.1990.
Bis(5'-adenosyl) tetraphosphate (Ap4A) phosphorylase II (P. Plateau, M. Fromant, J. M. Schmitter, J. M. Buhler, and S. Blanquet, J. Bacteriol. 171:6437-6445, 1989) was obtained in a homogeneous form through a 40,000-fold purification, starting from a Saccharomyces cerevisiae strain devoid of Ap4A phosphorylase I activity. The former enzyme behaves as a 36.8K monomer. As with Ap4A phosphorylase I, the addition of divalent cations is required for the expression of activity. Mn2+, Mg2+, and Ca2+ sustain phosphorolysis by the two enzymes, whereas Co2+ and Cd2+ stimulate only phosphorylase II activity. All bis(5'-nucleosidyl) tetraphosphates assayed (Ap4A, Ap4C, Ap4G, Ap4U, Gp4G, and Gp4U) are substrates of the two enzymes. However, Ap4A phosphorylase II shows a marked preference for A-containing substrates. The two enzymes catalyze adenosine 5'-phosphosulfate phosphorolysis or an exchange reaction between Pi and the beta-phosphate of any nucleoside diphosphate. They can also produce Ap4A at the expense of ATP and ADP. The gene (APA2) encoding Ap4A phosphorylase II was isolated and sequenced. The deduced amino acid sequence shares 60% identity with that of Ap4A phosphorylase I. Disruption of APA2 and/or APA1 shows that none of these genes is essential for the viability of Saccharomyces cerevisiae. The concentrations of all bis(5'-nucleosidyl) tetraphosphates are increased in an apa1 apa2 double mutant, as compared with the parental wild-type strain. The factor of increase is 5 to 50 times, depending on the nucleotide. This observation supports the conclusion that, in vivo, Ap4A phosphorylase II, like Ap4A phosphorylase I, participates in the catabolism rather than the synthesis of the bis(5'-nucleosidyl) tetraphosphates.
双（5'-腺苷）四磷酸（Ap4A）磷酸化酶II（P. 普拉泰奥、M. 弗罗曼、J. M. 施密特、J. M. 比勒和S. 布兰凯，《细菌学杂志》171:6437 - 6445，1989年）通过40000倍的纯化，从一株缺乏Ap4A磷酸化酶I活性的酿酒酵母菌株开始，以均一形式获得。前一种酶表现为36.8K的单体。与Ap4A磷酸化酶I一样，二价阳离子的添加是活性表达所必需的。Mn2 +、Mg2 +和Ca2 +维持这两种酶的磷酸解作用，而Co2 +和Cd2 +仅刺激磷酸化酶II的活性。所检测的所有双（5'-核苷基）四磷酸（Ap4A、Ap4C、Ap4G、Ap4U、Gp4G和Gp4U）都是这两种酶的底物。然而，Ap4A磷酸化酶II对含A的底物表现出明显的偏好。这两种酶催化腺苷5'-磷酸硫酸酯的磷酸解或Pi与任何核苷二磷酸的β-磷酸之间的交换反应。它们还可以以ATP和ADP为代价产生Ap4A。编码Ap4A磷酸化酶II的基因（APA2）被分离并测序。推导的氨基酸序列与Ap4A磷酸化酶I的氨基酸序列有60%的同一性。APA2和/或APA1的破坏表明这些基因中没有一个对酿酒酵母的生存能力是必需的。与亲本野生型菌株相比，apa1 apa2双突变体中所有双（5'-核苷基）四磷酸的浓度都增加了。增加的倍数为5至50倍，具体取决于核苷酸。这一观察结果支持这样的结论，即在体内，Ap4A磷酸化酶II与Ap4A磷酸化酶I一样，参与双（5'-核苷基）四磷酸的分解代谢而非合成。
