Tanaka Naoko, Hasan Zulfiqar, Hartog Aloysius F, van Herk Teunie, Wever Ron, Sanders Robert-Jan
Institute for Molecular Chemistry, University of Amsterdam, Nieuwe Achtergracht 129, The Netherlands.
Org Biomol Chem. 2003 Aug 21;1(16):2833-9. doi: 10.1039/b304012g.
Nonspecific acid phosphatases share a conserved active site with mammalian glucose-6-phosphatases (G6Pase). In this work we examined the kinetics of the phosphorylation of glucose and dephosphorylation of glucose-6-phosphate (G6P) catalysed by the acid phosphatases from Shigella flexneri (PhoN-Sf) and Salmonella enterica (PhoN-Se). PhoN-Sf is able to phosphorylate glucose regiospecifically to G6P, glucose-1-phosphate is not formed. The K(m) for glucose using pyrophosphate (PPi) as a phosphate donor is 5.3 mM at pH 6.0. This value is not significantly affected by pH in the pH region 4-6. The K(m) value for G6P by contrast is much lower (0.02 mM). Our experiments show these bacterial acid phosphatases form a good model for G6Pase. We also studied the phosphorylation of inosine to inosine monophosphate (IMP) using PPi as the phosphate donor. PhoN-Sf regiospecifically phosphorylates inosine to inosine-5'-monophosphate whereas PhoN-Se produces both 5'IMP and 3'IMP. The data show that during catalysis an activated phospho-enzyme intermediate is formed that is able to transfer its phosphate group to water, glucose or inosine. A general mechanism is presented of the phosphorylation and dephosphorylation reaction catalysed by the acid phosphatases. Considering the nature of the substrates that are phosphorylated it is likely that this class of enzyme is able to phosphorylate a wide range of hydroxy compounds.
非特异性酸性磷酸酶与哺乳动物葡萄糖-6-磷酸酶（G6Pase）共享一个保守的活性位点。在这项工作中，我们研究了福氏志贺菌（PhoN-Sf）和肠炎沙门氏菌（PhoN-Se）的酸性磷酸酶催化葡萄糖磷酸化和葡萄糖-6-磷酸（G6P）去磷酸化的动力学。PhoN-Sf能够将葡萄糖区域特异性地磷酸化为G6P，不会形成葡萄糖-1-磷酸。在pH 6.0时，以焦磷酸（PPi）作为磷酸盐供体时，葡萄糖的K(m)为5.3 mM。在4-6的pH范围内，该值不受pH的显著影响。相比之下，G6P的K(m)值要低得多（0.02 mM）。我们的实验表明，这些细菌酸性磷酸酶为G6Pase提供了一个很好的模型。我们还研究了以PPi作为磷酸盐供体时，肌苷磷酸化为肌苷单磷酸（IMP）的过程。PhoN-Sf将肌苷区域特异性地磷酸化为肌苷-5'-单磷酸，而PhoN-Se则产生5'IMP和3'IMP。数据表明，在催化过程中形成了一种活化的磷酸酶中间体，它能够将其磷酸基团转移到水、葡萄糖或肌苷上。本文提出了酸性磷酸酶催化的磷酸化和去磷酸化反应的一般机制。考虑到被磷酸化的底物的性质，这类酶可能能够磷酸化多种羟基化合物。
