Further Characterization of Aspartate Aminotransferase from Haloferax mediterranei: Pyridoxal Phosphate as Coenzyme and Inhibitor

Abstract

The enzyme aspartate aminotransferase has been isolated from the halophilic bacterium Haloferax mediterranei in its apoenzyme form. The interaction with its coenzyme (pyridoxal phosphate) has been investigated. For concentrations up to 0.05 mM, the incubation with pyridoxal phosphate reconstituted the active complex (holoenzyme) following a second order kinetic with a k2 of 5.2 min-1mM-1. This active complex showed a dissociation constant (Kd) of 7.8 x 10-6 M. For concentrations higher than 0.1 mM, pyridoxal phosphate produced an inactivation process with a complex second order kinetic. This inactivation is partially reverted by dialysis or by lysine treatment. Thus, after 80% of inactivation, 55% of the original activity is recovered by a long-time dialysis, and with 50 mM lysine also a partial reactivation (among 20-33%) is observed. The enzyme treated with 1 mM pyridoxal phosphate has a different behavior in Sepharose chromatography indicating that the modified enzyme presents a smaller size due to a conformational change. © 1995 Verlag der Zeitschrift für Naturforschung. All rights reserved.