Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine.
We report here the construction of a mutant version of Escherichia coli alkaline phosphatase (AP) in which the active site Ser was replaced by Thr (S102T), in order to investigate whether the enzyme can utilize Thr as the nucleophile and whether the rates of the critical steps in the mechanism are altered by the substitution. The mutant AP with Thr at position 102 exhibited an approximately 4000-fold decrease in k(cat) along with a small decrease in Km. The decrease in catalytic efficiency of approximately 2000-fold was a much smaller drop than that observed when Ala or Gly were substituted at position 102. The mechanism by which Thr can substitute for Ser in AP was further investigated by determining the X-ray structure of the S102T enzyme in the presence of the Pi (S102T_Pi), and after soaking the crystals with substrate (S102T_sub). In the S102T_Pi structure, the Pi was coordinated differently with its position shifted by 1.3 A compared to the structure of the wild-type enzyme in the presence of Pi. In the S102T_sub structure, a covalent Thr-Pi intermediate was observed, instead of the expected bound substrate. The stereochemistry of the phosphorus in the S102T_sub structure was inverted compared to the stereochemistry in the wild-type structure, as would be expected after the first step of a double in-line displacement mechanism. We conclude that the S102T mutation resulted in a shift in the rate-determining step in the mechanism allowing us to trap the covalent intermediate of the reaction in the crystal.
Wang
Jie
J
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Kantrowitz
Evan R
ER
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D012694:Serine / Q000737:chemistry*; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry*
D029968:Escherichia coli Proteins; D013912:Threonine; D012694:Serine; D000469:Alkaline Phosphatase