Wojciechowski Cheryl L, Cardia James P, Kantrowitz Evan R
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Protein Sci. 2002 Apr;11(4):903-11. doi: 10.1110/ps.4260102.
The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.
嗜热栖热菌编码一个与几个已知碱性磷酸酶（AP）基因具有序列相似性的基因。分离出该假定基因，并在大肠杆菌中表达了相应的蛋白质，表达时带有和不带有预测的信号序列。重组蛋白对底物对硝基苯磷酸酯表现出磷酸酶活性，在25℃测定时，pH最佳值为8.0时，催化常数（k(cat)）为16 s(-1)，米氏常数（K(m)）为175 microM。嗜热栖热菌磷酸酶活性在高温下增加，在65℃时达到最大催化常数100 s(-1)，米氏常数为93 microM。活性在65℃下>24小时稳定，在90℃下5小时稳定。磷酸酶活性依赖于二价金属离子，特别是Co(II)和Mg(II)。圆二色光谱表明，添加这些金属后酶获得二级结构。锌是已知AP活性所需的最常见二价金属离子，在浓度高于0.3摩尔锌：1摩尔单体时，显示出抑制嗜热栖热菌磷酸酶。在存在0.1 mM EDTA时所有活性都被消除。与大肠杆菌AP相比，嗜热栖热菌AP的一级序列有28%的同一性。基于结构模型，除了大肠杆菌AP镁结合位点附近的两个残基外，活性位点是可叠加的，这两个残基分别对应嗜热栖热菌AP中的组氨酸和色氨酸，即大肠杆菌编号中的D153和K328。蔗糖密度梯度沉降实验表明，该蛋白质以几种四级形式存在，主要是八聚体。
