Affordable Access

Analysis of electrostatic interactions in a low molecular weight tyrosine phosphatase and an acid phosphatase/phytase using NMR spectroscopy

Purdue University
Publication Date
  • Chemistry
  • Biochemistry
  • Biology


Electrostatic interactions in bovine low molecular weight protein tyrosine phosphatase (BPTP) were studied by site-directed mutagenesis, 1H NMR spectroscopy, and theoretical calculations. Amino acid residue His-72 is located near the active site and has a pKa of 9.2 while His-66 is ∼20 Å away and has a pKa of 8.3. The reasons for these unusually high pKaS were examined by mutating amino acid residues near each histidine, and measuring the effect on the corresponding pKa using NMR spectroscopy. The electrostatic interactions that affected the histidine pKaS included negatively charged groups, hydrogen bonding groups, and an aromatic group coplanar to the imidazole ring. Computer simulations using the linear Poisson-Boltzmann equation adequately predicted the relative changes in pKaS and were consistent with the experimental values. Similar experiments were used to analyze the electrostatic interactions in a significantly larger acid phosphatase/phytase from Escherichia coli (EcAP, 45 kDa) with eight histidines. The His-17 residue is the active site nucleophile and apparently has an unusually low pKa in order to account for pH optimum of 2.5 for EcAP phosphatase activity. Three of eight histidine mutants (H17N, H303A, and H250A) exhibited significant decreases in phosphatase and phytase activity. Most of the histidines in EcAP had unperturbed pKaS (∼6.5–7). However, His-250 did not appear to titrate in the pH range of 1–10, and the pKa of His-17 could not be determined. The His-303 pH titration curve in wild type EcAP had an unusual inflection at low pH (pKa ∼ 3) indicating that a structural change at the active site affects His-303 at low pH, and His-17 and Asp-304 appear to be involved. ^

There are no comments yet on this publication. Be the first to share your thoughts.


Seen <100 times