Mutations at positions 153 and 328 in Escherichia coli alkaline phosphatase provide insight towards the structure and function of mammalian and yeast alkaline phosphatases.
Mutations at positions 153 and 328 in Escherichia coli alkaline phosphatase provide insight towards the structure and function of mammalian and yeast alkaline phosphatases.
复制标题
大肠杆菌碱性磷酸酶中 153 和 328 位的突变提供了对哺乳动物和酵母碱性磷酸酶的结构和功能的深入了解。
DOI：
10.1006/jmbi.1995.0576
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Kantrowitz,ER
中科院分区：
文献类型：
--
作者：
Murphy,JE;Tibbitts,TT;Kantrowitz,ER
In order to understand some of the differences between human placental, human,Saccaromyces cerevisiaeandEscherichia colialkaline phosphatases in specific activity, activation by magnesium, and pHversusactivity profiles, the X-ray crystal structures of three mutantE. colialkaline phosphatases have been determined. The aligned sequences of alkaline phosphatases from mammalian, yeast andE. colishow 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 theE. colienzyme, 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 Å with a workingR-factor of 0.181 and a freeR-factor of 0.249. The DM structure was determined to a resolution of 2.5 Å with a workingR-factor of 0.166 and a free R-factor of 0.249. The DM structure was determined to a resolution of 2.5 Å with a workingR-factor of 0.166 and a freeR-factor of 0.233. The structure of the D135H enzyme, which has been reported to a resolution of 2.4 Å, has been re-refined using cross-validation to a workingR-factor of 0.179 and a freeR-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.
