Abstract:Phosphoenolpyvate carboxykinase (PEPCK, EC 4. 1. 1. 49) from pineapple leaf was puri-fied by the procedures including amm-onium sulfate precipitation, chromatography on Sephadex G-200 and DEAE-cellulose. The purified enzyme showed a homo-genous band oh polyacry-lamide gel electrophoretogram. The conformation of PEPCK protein was studied by ultraviolet absorption difference spectrum and fluorescence spectrum. The binding of enzyme with substrate and Mn2+ resulted in the changes of ultraviolet absorption difference spectra at peak sharpness, height and orientation. Substrate OAA induced a broad negative band at 268-280nm, but two negative bands at 242. 5nm and 273. 5nm were observed when another substrate ATP was bound alone to PEPCK. By addition of both ATP and OAA, the spectrum showed two positive peaks at 252nm and 268nm. The changes of spectra orientation and shift of wavelength were found in the presence of Mrt2+ either with ATP or ATP+OAA. The enzyme protein solution showed a maximum fluorescence emission at 323nm, OAA markedly enhanced the emission, whereas ATP or ATP + Mn2+ declined the fluorescence. The combination of OAA+ATP+Mn2+ with PEPCK induced the further decrease of fluorescence. These results suggest that the alternative conformation states was generated during the binding of PEPCK to its substrate and metal ion. The difference conformation state between PEPCK + OAA and PEPCK + ATP indicated that these two compounds were bound to different sites of PEPCK protein. Mn2+ might bind together with ATP on the same site of enzyme. The changes of spectra orientation caused by Mn2+may be explained as a beneficial conformation for catalysis of PEPCK.
