Isolation And Some Properties Of 6-Phosphogluconate Dehydrogenase From Bacillus Subtilis

6-Phosphogluconate dehydrogenase(6-PGADase, EC 1.1.1.44) was isolated by homogenate, ammunium sulfate fractionation, DEAE-sepharose chromatography, Blue-sepharose Affinity chromatography and gel filtration with Sephadex G-200 from bacillus subtilis, and some properties of the enzyme had been studied. A 113.8-fold purification was obtained with a 8.2% yield. The purified enzyme moved as a single electrophoretic band in PAGE . The molecule weight of the 6-PGADase is 110000Da,which is measured by AKTA FPLC and the molecule weight of subunit is 51000Da, which is measured by SDS-PAGE. Therefore one6-PGADase has two same subunits. Dynamic analysis showed that there is no copp eration effect between subunits. It's pI is 5.2 as determined with IEF. Amino acid composition analysis showed that one subunit of 6-Phosphogluconate dehydrogenase has about 480 amino acids and there are plentiful of Ala, Asp, Leu, Ser, Glu, Thr, Phe, Lys in it. It was demonstrated that the enzyme has 43.7% a -helix, 26.9% β -pleated,29.4%random coil by the analysis of circular dichroism spectrum.Dynamic analysis showed that the optimum of the activity was found at about pH8.0 and at 30℃ ,with a Km value of 19.8μmol/L for NADP and 22.6μ mol/L for 6-phosphogluconic acid. The 6-PGADase is activated by Mg2+, Mn2+ and Zn2+,while the enzyme is inhibited by cations of Cu2+ and Fe3+. Na+, K + and anions such as Cl-, NO3-, SO42- have no effect on the 6-PGADase in concentration ranging from 5mM to 50mM. PMSF, TNBS, NBS, DTT and BrAc are used to modify the 6-PGADase, and the results illustrate that Ser, Thr, Lys and His are critical for the activity of the enzyme.Kinetic study shows that NADP+ is the only valid coenzyme and that one substrate have no effect on the the other substrates' bind with the Enzyme. Products are competitive inhibitors, NADP+/NADPH and 6-phosphogluconate /ribulose-5-phosphate pairs are competitive whatever the concentration of the other substrates but noncompetitive versus the other substrates. Therefore the Kinetic mechanism of the 6-phosphogluconate dehydrogenase is Haldance-Laidler-Socquet random mechanism.