Murphy J E, Tibbitts T T, Kantrowitz E R
Boston College, Department of Chemistry, Merkert Chemistry Center, Chestnut Hill, MA 02167-3860, USA.
J Mol Biol. 1995 Nov 3;253(4):604-17. doi: 10.1006/jmbi.1995.0576.
In order to understand some of the differences between human placental, human, Saccharomyces cerevisiae and Escherichia coli alkaline phosphatases in specific activity, activation by magnesium, and pH versus activity profiles, the X-ray crystal structures of three mutant E. coli alkaline phosphatases have been determined. The aligned sequences of alkaline phosphatases from mammalian, yeast and E. coli show that 25 to 30% of the amino acids are absolutely conserved and the active site residues are completely conserved with the exception of residues 153, 328 and 155. The bacterial enzyme has a salt-bridge, Asp153/Lys328, near the third metal binding site which, based on sequence homology, is apparently absent in the yeast and mammalian enzymes. The human enzymes have histidine at positions 153 and 328, and the yeast enzyme has histidine at position 328. In the E. coli enzyme, Asp153 was replaced by histidine (D153H), Lys328 was replaced by histidine (K328H), and a double mutant (DM) was constructed containing both mutations. The structure of the K328H enzyme was refined using cross-validation to a resolution of 2.3 A with a working R-factor of 0.181 and a free R-factor of 0.249. The DM structure was determined to a resolution of 2.5 A with a working R-factor of 0.166 and a free R-factor of 0.233. The structure of the D135H enzyme, which has been reported to a resolution of 2.4 A, has been re-refined using cross-validation to a working R-factor of 0.179 and a free R-factor of 0.239 for controlled comparisons with the two new structures. In all three structures the most significant changes are related to the bound phosphate inhibitor and the identity of the metal ion in the third binding site. The changes in the position of the phosphate group and the alterations at the third metal binding site indicate the structural basis for the variations in the steady-state kinetic parameters previously reported for these enzymes.
为了了解人胎盘碱性磷酸酶、人碱性磷酸酶、酿酒酵母碱性磷酸酶和大肠杆菌碱性磷酸酶在比活性、镁激活作用以及pH值与活性曲线方面的一些差异，已测定了三种突变型大肠杆菌碱性磷酸酶的X射线晶体结构。来自哺乳动物、酵母和大肠杆菌的碱性磷酸酶的比对序列表明，25%至30%的氨基酸是完全保守的，除了153、328和155位残基外，活性位点残基是完全保守的。细菌酶在第三个金属结合位点附近有一个盐桥Asp153/Lys328，基于序列同源性，酵母和哺乳动物酶中显然没有这个盐桥。人酶在153和328位有组氨酸，酵母酶在328位有组氨酸。在大肠杆菌酶中，Asp153被组氨酸取代（D153H），Lys328被组氨酸取代（K328H），并构建了包含这两种突变的双突变体（DM）。使用交叉验证将K328H酶的结构精修至2.3 Å的分辨率，工作R因子为0.181，自由R因子为0.249。DM结构的分辨率为2.5 Å，工作R因子为0.166，自由R因子为0.233。已报道分辨率为2.4 Å的D135H酶的结构，已使用交叉验证重新精修，工作R因子为0.179，自由R因子为0.239，以便与两个新结构进行对照比较。在所有三种结构中，最显著的变化与结合的磷酸抑制剂以及第三个结合位点中的金属离子的特性有关。磷酸基团位置的变化以及第三个金属结合位点的改变表明了先前报道的这些酶的稳态动力学参数变化的结构基础。
