Thompson J, Chassy B M
J Bacteriol. 1983 Oct;156(1):70-80. doi: 10.1128/jb.156.1.70-80.1983.
An intracellular hexose 6-phosphate:phosphohydrolase (EC 3.1.3.2) has been purified from Streptococcus lactis K1. Polyacrylamide disc gel electrophoresis of the purified enzyme revealed one major activity staining protein and one minor inactive band. The Mr determined by gel permeation chromatography was 36,500, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single polypeptide of apparent Mr 60,000. The enzyme exhibited a marked preference for hexose 6-phosphates, and the rate of substrate hydrolysis (at 5 mM concentration) decreased in the order, galactose 6-phosphate greater than 2-deoxy-D-glucose 6-phosphate greater than fructose 6-phosphate greater than mannose 6-phosphate greater than glucose 6-phosphate. Hexose 1-phosphates, p-nitrophenylphosphate, pyrophosphate, and nucleotides were not hydrolyzed at a significant rate. In addition, the glycolytic intermediates comprising the intracellular phosphoenolpyruvate potential in the starved cells (phosphoenolpyruvate and 2- and 3-phosphoglyceric acids) were not substrates for the phosphatase. Throughout the isolation, the hexose 6-phosphate:phosphohydrolase was stabilized by Mn2+ ion, and the purified enzyme was dependent upon Mn2+, Mg2+, Fe2+, or Co2+ for activation. Other divalent metal ions including Pb2+, Cu2+, Zn2+, Cd2+, Ca2+, Ba2+, Sr2+, and Ni2+ were unable to activate the enzyme, and the first four cations were potent inhibitors. Enzymatic hydrolysis of 2-deoxy-D-glucose 6-phosphate was inhibited by fluoride when Mg2+ was included in the assay, but only slight inhibition occurred in the presence of Mn2+, Fe2+, or Co2+. The inhibitory effect of Mg2+ plus fluoride was specifically and completely reversed by Fe2+ ion. The hexose 6-phosphate:phosphohydrolase catalyzes the in vivo hydrolysis of 2-deoxy-D-glucose 6-phosphate in stage II of the phosphoenolpyruvate-dependent futile cycle in S. lactis (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982).
已从乳酸链球菌K1中纯化出一种细胞内己糖6 - 磷酸：磷酸水解酶（EC 3.1.3.2）。纯化酶的聚丙烯酰胺圆盘凝胶电泳显示出一条主要的活性染色蛋白带和一条次要的无活性带。通过凝胶渗透色谱法测定的相对分子质量为36,500，但十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示出一条表观相对分子质量为60,000的单一多肽。该酶对己糖6 - 磷酸表现出明显的偏好，底物水解速率（在5 mM浓度下）按以下顺序降低：6 - 磷酸半乳糖＞6 - 磷酸2 - 脱氧 - D - 葡萄糖＞6 - 磷酸果糖＞6 - 磷酸甘露糖＞6 - 磷酸葡萄糖。1 - 磷酸己糖、对硝基苯磷酸、焦磷酸和核苷酸均未以显著速率被水解。此外，饥饿细胞中构成细胞内磷酸烯醇丙酮酸势的糖酵解中间产物（磷酸烯醇丙酮酸以及磷酸甘油酸和3 - 磷酸甘油酸）不是该磷酸酶的底物。在整个分离过程中，己糖6 - 磷酸：磷酸水解酶由Mn2 +离子稳定，纯化后的酶依赖于Mn2 +、Mg2 +、Fe2 +或Co2 +进行激活。包括Pb2 +、Cu2 +、Zn2 +、Cd2 +、Ca2 +、Ba2 +、Sr2 +和Ni2 +在内的其他二价金属离子无法激活该酶，前四种阳离子是强效抑制剂。当测定中含有Mg2 +时，6 - 磷酸2 - 脱氧 - D - 葡萄糖的酶促水解受到氟化物的抑制，但在存在Mn2 +、Fe2 +或Co2 +时仅发生轻微抑制。Mg2 +加氟化物的抑制作用可被Fe2 +离子特异性且完全逆转。己糖6 - 磷酸：磷酸水解酶催化乳酸链球菌中磷酸烯醇丙酮酸依赖性无效循环第二阶段6 - 磷酸2 - 脱氧 - D - 葡萄糖的体内水解（J. 汤普森和B. M. 查西，《细菌学杂志》151:1454 - 1465，1982）。
