Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine

Abstract

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.

Figure 1.

(A) Stereoview of the active site of the S102T_P_i structure. Shown is the 2F _o –F _c electron density map for the A chain (1.2 σ). The side chain of the Thr102 are in the χ = −60° conformation). Water molecules are shown as red spheres and zinc and magnesium atoms as large dark blue and cyan spheres. Dashed lines represent hydrogen bonds. (B) Stereoview of the 2F _o –F _c electron density map for the B chain of S102T_sub (1.2 σ). (C) Comparison of Arg166 and P_i in the active sites of the S102T_P_i structure (elemental colors, thick lines) and the WT_P_i structure (PDB code 1ED8; Stec et al. 2000; green, thin) in the A chain and in the B chain (D). Ligands around the metals are not shown. (E) Active site of the S102T_sub structure. The Arg166 and metals are overlaid on the 2F _o –F _c electron density map (blue) shown contoured at 1.2 σ. The ThP102 is overlaid on the F _o –F _c electron density map (magenta) shown contoured at 1.5 σ. (F) Comparison of P_i and Ser102 (ThP102) in the active site of the S102T_sub structure (elemental colors, thick lines) and the WT_P_i (green, thin) structure. Ligands around the metals are not shown. Figures were prepared using MOLSCRIPT (Kraulis 1991).