Isotope exchange studies on the Escherichia coli selenophosphate synthetase mechanism
Isotope exchange studies on the Escherichia coli selenophosphate synthetase mechanism
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DOI：
10.1073/pnas.95.5.2180
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发表时间：
1998-03-03
影响因子：
11.1
通讯作者：
Stadtman, TC
中科院分区：
文献类型：
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作者：
Walker, H;Ferretti, JA;Stadtman, TC
硒磷酸合成酶是大肠杆菌selD基因产物，是一种37 kda的蛋白质，催化ATP和硒化物合成硒磷酸。在没有硒化物的情况下，ATP在一个非常缓慢的部分反应中被定量地转化为AMP和两个正磷酸盐。在最终的硒化磷酸产物中，磷酸键能的守恒是由它磷酸化醇和胺形成o -磷酰和n -磷酰衍生物的能力表明的。为了进一步探索硒磷酸合成酶的作用机制，我们用[8-C-14]ADP或[8-C-14]AMP和未标记的ATP进行了同位素交换研究，并对富集(H2O)-O-18的反应混合物进行了P-31 NMR分析。在没有硒化物的情况下，观察到酶催化ADP与ATP缓慢交换，这表明存在磷酸化酶，进一步支持了ADP在反应中的中介作用。在这些条件下，ADP缓慢地转化为AMP。在整个硒依赖反应中，(H2O)-O-18将O-18完全结合到正磷酸盐中，表明酶结合ADP的β -磷酸基受到水的攻击，正磷酸盐被释放并形成AMP。基于这些结果和酶无法催化标记AMP与ATP的交换。可以排除先前假设的焦磷酸化酶中间体的存在。
Selenophosphate synthetase, the Escherichia coli selD gene product, is a 37-kDa protein that catalyzes the synthesis of selenophosphate from ATP and selenide, In the absence of selenide, ATP is converted quantitatively to AMP and two orthophosphates in a very slow partial reaction, A monophosphorylated enzyme derivative containing the gamma-phosphoryl group of ATP has been implicated as an intermediate from the results of positional isotope exchange studies, Conservation of the phosphate bond energy in the final selenophosphate product is indicated by its ability to phosphorylate alcohols and amines to form O-phosphoryl- and N-phosphoryl-derivatives. To further probe the mechanism of action of selenophosphate synthetase, isotope exchange studies with [8-C-14]ADP or [8-C-14]AMP and unlabeled ATP were carried out, and P-31 NMR analysis of reaction mixtures enriched in (H2O)-O-18 was performed, A slow enzyme-catalyzed exchange of ADP with ATP observed in the absence of selenide implies the existence of a phosphorylated enzyme and further supports an intermediary role of ADP in the reaction, Under these conditions ADP is slowly converted to AMP. Incorporation of O-18 from (H2O)-O-18 exclusively into orthophosphate in the overall selenide-dependent reaction indicates that the beta-phosphoryl group of the enzyme-bound ADP is attacked by water with liberation of orthophosphate and formation of AMP, Based on these results and the failure of the enzyme to catalyze an exchange of labeled AMP with ATP, the existence of a pyrophosphorylated enzyme intermediate that was postulated earlier can be excluded.