Probing the function of conserved residues in the serine/threonine phosphatase PP2Calpha

Biochemistry. 2003 Jul 22;42(28):8513-21. doi: 10.1021/bi034074+.

Abstract

Human PP2Calpha is a metal-dependent phosphoserine/phosphothreonine protein phosphatase and is the representative member of the large PPM family. The X-ray structure of human PP2Calpha has revealed an active site containing a dinuclear metal ion center that is coordinated by several invariant carboxylate residues. However, direct evidence for the catalytic function of these and other active-site residues has not been established. Using site-directed mutagenesis and enzyme kinetic analyses, we probed the roles of conserved active-site amino acids within PP2Calpha. Asp-60 bridges metals M1 and M2, and Asp-239 coordinates metal M2, both of which were replaced individually to asparagine residues. These point mutations resulted in >or=1000-fold decrease in k(cat) and >or=30-fold increase in K(m) value for Mn(2+). Mutation of Asp-282 to asparagine caused a 100-fold decrease in k(cat), but no significant effect on K(m) values for metal and substrate, consistent with Asp-282 activating a metal-bound water nucleophile. Mutants T128A, E37Q, D38N, and H40A displayed little or no alterations on k(cat) and K(m) values for substrate or metal ion (Mn(2+)). Analysis of H62Q and R33A yielded k(cat) values that were 20- and 2-fold lower than wild-type, respectively. The mutant R33A showed a 8-fold higher K(m) for substrate, while the K(m) observed with H62Q was unaffected. A pH-rate profile of the H62Q mutant showed loss of the ionization that must be protonated for activity. Brönsted analysis of substrate leaving group pK(a) values for H62Q indicated a greater dependency (slope -0.84) on leaving group pK(a) in comparison to wild-type (slope -0.33). These data provide strong evidence that His-62 acts as a general acid during the cleavage of the P-O bond.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Conserved Sequence
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Ligands
  • Mutagenesis, Site-Directed
  • Phosphoprotein Phosphatases / chemistry*
  • Phosphoprotein Phosphatases / genetics
  • Phosphoprotein Phosphatases / isolation & purification
  • Phosphoprotein Phosphatases / metabolism*
  • Protein Conformation
  • Protein Phosphatase 2C
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • Ligands
  • Recombinant Proteins
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2C