Mn(2+) in D-Glucosaminate Dehydratase from Pseudomonas fluorescens

D-Glucosaminate (D-GlcNA) dehydratase from Pseudomonas fluorescens was inhibited stoichiometrically by metal-chelating agents (EDTA, 8-hydroxyquinoline-5-sulfonic acid, α,α'-dipyridyl and o-phenan-throline). The activity of EDTA-treated enzyme was restored by incubation with Mn(2+) (0.4mM) or Ca(2+) (2mM) in the presence of pyridoxal 5'-phosphate (PLP, 0.2mM) in veronal buffer (VB, 40 mM, pH 8) at 37°C for 30 min. The atomic absorption spectrum of the native enzyme showed that the enzyme contained 1 mol of Mn(2+) per mole of enzyme. Although the EDTA-treated enzyme was unstable at 4°C, addition of Mn(2+) and PLP to the solution of the EDTA-treated enzyme prevented the inactivation. The Km of the restored enzyme for D-GlcNA was somewhat lower than that of the original enzyme. However, the Km for PLP increased 14-fold. These results suggest that D-GlcNA dehydratase contains Mn(2+) near the PLP-binding site, and the metal ion appears to stabilize the structure of the active site.