The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site-specific mutagenesis

The proposed double in-line displacement mechanism of Escherichia coli alka line phosphatase (AP) involving two-metal ion catalysis is based on NMR spe ctroscopic and X-ray crystallographic studies. This mechanism is further su pported by the X-ray crystal structures of the covalent phospho-enzyme inte rmediate of the H331Q mutant AP and of the transition state complex between the mild-type enzyme and vanadate, a transition state analog, Kinetic and structural studies on several genetically engineered versions of AP illustr ate the overall importance of the active site's metal geometry, hydrogen bo nding network and electrostatic potential in the catalytic mechanism, (C) 1 999 Federation of European Biochemical Societies.