Mn^<2+> in D-Glucosaminate Dehydratase from Pseudomonas fluorescens

概要

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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+> (0.4 mM) or Ca^<2+>(2 mM) in the presence of pyridoxal 5'-phosphate (PLP, 0.2 mM) in veronal buffer (VB, 40 mM, pH 8) at 37℃ 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℃, addition of Mn^<2+> and PLP to the solution of the EDTA-treated enzyme prevented the inactivation. The K_m of the restored enzyme for D-GlcNA was somewhat lower than that of the original enzyme. However, the K_m 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.
社団法人日本農芸化学会の論文
1993-07-23