FURTHER CHARACTERIZATION OF ASPARTATE-AMINOTRANSFERASE FROM HALOFERAX-MEDITERRANEI - PYRIDOXAL-PHOSPHATE AS COENZYME AND INHIBITOR

The enzyme aspartate aminotransferase has been isolated from the halop hilic bacterium Haloferax mediterranei in its apoenzyme form. The inte raction with its coenzyme (pyridoxal phosphate) has been investigated. For concentrations up to 0.05 mM, the incubation with pyridoxal phosp hate reconstituted the active complex (holoenzyme) following a second order kinetic with a k(2) Of 5.2 min(-1)mM(-1). This active complex sh owed a dissociation constant (K-d) of 7.5 x 10(-6) M. For concentratio ns higher than 0.1 mM: pyridoxal phosphate produced an inactivation pr ocess with a complex second order kinetic. This inactivation is partia lly reverted by dialysis or by lysine treatment. Thus, after 80% of in activation, 55% of the original activity is recovered by a long-time d ialysis, and with 50 mM lysine also a partial reactivation (among 28-3 3%) is observed. The enzyme treated with 1 mM pyridoxal phosphate has a different behavior in Sepharose chromatography indicating that the m odified enzyme presents a smaller size due to a conformational change.