来自酵母的二腺苷5′,5‴-P1,P4-四磷酸α,β-磷酸化酶可支持核苷二磷酸-磷酸交换。

Diadenosine 5',5'''-P1, P4-tetraphosphate alpha, beta-phosphorylase from yeast supports nucleoside diphosphate-phosphate exchange.

作者信息

Guranowski A, Blanquet S

出版信息

J Biol Chem. 1986 May 5;261(13):5943-6.

Abstract

Homogeneous diadenosine 5',5'''-P1,P4-tetraphosphate alpha, beta-phosphorylase (Ap4A-phosphorylase), the enzyme recently found in yeast (Guranowski, A., and Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547) catalyzes an exchange reaction between the beta-phosphate of nucleoside diphosphate (NDP) and orthophosphate from the medium (Pi). The common purine and pyrimidine ribonucleoside diphosphates as well as ADP analogs modified either in aglycone, sugar, or at the anhydride bond beta-position are substrates. The Km and rate values for the NDP-Pi exchange reaction were estimated at pH optima. These optima are 6.5 for UDP, 7.0 for ADP or CDP, and 8.0 for GDP. In the presence of 10 mM K2HPO4, 0.1 mM EDTA, and 100 mM Hepes/KOH (pH 7.0), the Km for ADP is 0.7 mM with a rate constant at saturating ADP of 96 s-1. The Km value for orthophosphate is 2 mM. In the NDP-Pi exchange reaction, phosphate can be substituted with arsenate and apparent arsenolysis of NDPs yields corresponding nucleoside monophosphates. The same pH optimum of 6.5 is found for arsenolysis of ADP, GDP, and CDP. Whereas the Ap4A phosphorylase sulfhydryl groups are essential for catalyzing the Ap4A phosphorolysis, the NDP-Pi exchange reactions, and the arsenolysis of NDPs, the divalent metal ions (Mg2+, Mn2+, Ca2+, Co2+, and Cd2+), which had been shown to be essential cofactors of the former reaction, are not required for the two latter ones. Used at concentrations which are optimum for Ap4A phosphorolysis, the cations (particularly Mg2+ and Cd2+) inhibit the NDP-Pi exchange and the arsenolysis of NDPs. Interestingly, the Ap4A phosphorylase exhibits higher specificity for adenosine 5'-phosphosulfate (APS) than for any other NDP tested. The V/Km ratio is almost 5-fold higher with APS than with ADP. However, in the presence of orthophosphate, the APS is irreversibly converted to ADP. Thus, the enzyme displays a property already attributed to ADP-sulfurylase (EC 2.7.7.5), (Grunberg-Manago, M., Del Campillo-Campbell, A., Dondon, L., and Michelson, A. M. (1966) Biochim. Biophys. Acta 123, 1-16; Nicholls, R. G. (1977) Biochem. J. 165, 149-155).

摘要

均匀二腺苷5',5'''-P1,P4-四磷酸α,β-磷酸化酶（Ap4A-磷酸化酶），这种最近在酵母中发现的酶（古兰诺夫斯基，A.，和布兰凯，S.（1985年）《生物化学杂志》260，3542 - 3547）催化核苷二磷酸（NDP）的β-磷酸与介质中的正磷酸盐（Pi）之间的交换反应。常见的嘌呤和嘧啶核糖核苷二磷酸以及在糖苷配基、糖或酸酐键β-位修饰的ADP类似物都是底物。在最适pH值下估算了NDP - Pi交换反应的Km值和速率值。这些最适pH值对于UDP是6.5，对于ADP或CDP是7.0，对于GDP是8.0。在含有10 mM K2HPO4、0.1 mM EDTA和100 mM Hepes/KOH（pH 7.0）的条件下，ADP的Km值为0.7 mM，饱和ADP时的速率常数为96 s-1。正磷酸盐的Km值为2 mM。在NDP - Pi交换反应中，磷酸盐可以被砷酸盐替代，NDP的明显砷解会产生相应的核苷单磷酸。ADP、GDP和CDP的砷解反应也发现相同的最适pH值为6.5。虽然Ap4A磷酸化酶的巯基对于催化Ap4A磷酸解、NDP - Pi交换反应以及NDP的砷解是必不可少的，但二价金属离子（Mg2+、Mn2+、Ca2+、Co2+和Cd2+），已被证明是前一个反应的必需辅因子，而后两个反应并不需要。以对Ap4A磷酸解最适宜的浓度使用时，阳离子（特别是Mg2+和Cd2+）会抑制NDP - Pi交换和NDP的砷解。有趣的是，Ap4A磷酸化酶对腺苷5'-磷酸硫酸酯（APS）的特异性高于所测试的任何其他NDP。APS的V/Km比值几乎比ADP高5倍。然而，在正磷酸盐存在的情况下，APS会不可逆地转化为ADP。因此，该酶表现出一种已归因于ADP - 硫酸化酶（EC 2.7.7.5）的特性，（格伦贝格 - 马纳戈，M.，德尔坎皮洛 - 坎贝尔，A.，东东，L.，和米切尔森，A. M.（1966年）《生物化学与生物物理学报》123，1 - 16；尼科尔斯，R. G.（1977年）《生物化学杂志》165，149 - 155）。