Effect of ionic strength and oxidation on the P-loop conformation of the protein tyrosine phosphatase-like phytase, PhyAsr.
The protein tyrosine phosphatase (PTP)-like phytase, PhyAsr, from Selenomonas ruminantium is a novel member of the PTP superfamily, and the only described member that hydrolyzes myo-inositol-1,2,3,4,5,6-hexakisphosphate. In addition to the unique substrate specificity of PhyAsr, the phosphate-binding loop (P-loop) has been reported to undergo a conformational change from an open (inactive) to a closed (active) conformation upon ligand binding at low ionic strength. At high ionic strengths, the P-loop was observed in the closed, active conformation in both the presence and absence of ligand. To test whether the P-loop movement can be induced by changes in ionic strength, we examined the effect that ionic strength has on the catalytic efficiency of PhyAsr, and determined the structure of the enzyme at several ionic strengths. The catalytic efficiency of PhyAsr is highly sensitive to ionic strength, with a seven-fold increase in k(cat)/K(m) and a ninefold decrease in K(m) when the ionic strength is increased from 100 to 500 mm. Surprisingly, the P-loop is observed in the catalytically competent conformation at all ionic strengths, despite the absence of a ligand. Here we provide structural evidence that the ionic strength dependence of PhyAsr and the conformational change in the P-loop are not linked. Furthermore, we demonstrate that the previously reported P-loop conformational change is a result of irreversible oxidation of the active site thiolate. Finally, we rationalize the observed P-loop conformational changes observed in all oxidized PTP structures.
Department of Chemistry and Biochemistry, University of Lethbridge, Canada.
Robert J
RJ
Gruninger
L Brent
LB
Selinger
Steven C
SC
Mosimann
D010832:6-Phytase / Q000737:chemistry*; D002384:Catalysis; D007700:Kinetics; D008958:Models, Molecular; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry*
D010832:6-Phytase; D017027:Protein Tyrosine Phosphatases