Gerlt J A, Whitman G J
J Biol Chem. 1975 Jul 10;250(13):5053-8.
A phosphohydrolase from Enterobacter aerogenes which hydrolyzes phosphate mono- and diesters has been purified approximately 50-fold to apparent homoeneity and crystallized. The enzyme is produced when the bacteria utilize phosphate diesters as sole phosphorus source. From sedimentation equilibrium experiments the molecular weight of the native enzyme is 173,000; from sodium dodecyl sulfate polyacrylamide gel electrophoresis the subunit molecular weight is 29,000, indicating that the enzyme is hexameric. The hydrolytic activity of the enzyme using both mono- and diesters is maximal at pH 5; THE Km of the enzyme for bis-p-nitrophenyl phosphate is constant from pH 5 to 8.5 whereas that for p-nitrophenyl phosphate increases about 40-fold as the pH increases over the same range. The phosphodiesterase activity is not inhibited by chelating agents but is inhibited by several divalent metal ions. 31-P NMR spectroscopy was used to identify the hydrolysis products of glycoside cyclic phosphates. The enzyme-catalyzed hydrolysis of methyl beta-D-ribofuranoside cyclic 3:5-phosphate yields exclusively the 5-phosphate whereas that of adenosine 3:5-monophosphate yields a 4:1 mixture of 3- and 5- AMP.
一种来自产气肠杆菌的磷酸水解酶，可水解磷酸单酯和二酯，已被纯化约50倍至表观均一，并进行了结晶。当细菌利用磷酸二酯作为唯一磷源时会产生这种酶。通过沉降平衡实验，天然酶的分子量为173,000；通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳，亚基分子量为29,000，表明该酶是六聚体。该酶对单酯和二酯的水解活性在pH 5时最大；该酶对双对硝基苯磷酸酯的Km在pH 5至8.5范围内保持恒定，而对硝基苯磷酸酯的Km在相同pH范围内随pH升高增加约40倍。磷酸二酯酶活性不受螯合剂抑制，但受几种二价金属离子抑制。采用31-P核磁共振光谱法鉴定糖苷环磷酸酯的水解产物。该酶催化β-D-呋喃核糖苷环3:5-磷酸甲酯的水解仅产生5-磷酸酯，而腺苷3:5-单磷酸酯的水解产生3-和5-AMP的4:1混合物。
