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The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site‐specific mutagenesis
Author(s) -
Holtz Kathleen M,
Kantrowitz Evan R
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01448-9
Subject(s) - alkaline phosphatase , mutagenesis , chemistry , site directed mutagenesis , mechanism (biology) , crystallography , biochemistry , mutation , enzyme , mutant , gene , physics , quantum mechanics
The proposed double in‐line displacement mechanism of Escherichia coli alkaline phosphatase (AP) involving two‐metal ion catalysis is based on NMR spectroscopic and X‐ray crystallographic studies. This mechanism is further supported by the X‐ray crystal structures of the covalent phospho‐enzyme intermediate of the H331Q mutant AP and of the transition state complex between the wild‐type enzyme and vanadate, a transition state analog. Kinetic and structural studies on several genetically engineered versions of AP illustrate the overall importance of the active site's metal geometry, hydrogen bonding network and electrostatic potential in the catalytic mechanism.