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

• Published in: Zeitschrift für Naturforschung C. A journal of biosciences Vol. 50; no. 3; pp. 241 - 247
• Main Authors: García-Muriana, Francisco J., Alvarez-Ossorioa, María C., Sánchez-Garcés, María M., Rosa, F. de la, Relimpio, Angel M.
• Format: Journal Article
• Language: English
• Published: Verlag der Zeitschrift für Naturforschung 01-04-1995
• Subjects: Activity and Stability; Aspartate Aminotransferase; Haloferax mediterranei; Halophilic Bacteria; PLP Effects
• ISSN: 0939-5075; 1865-7125
• DOI: 10.1515/znc-1995-3-413

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 mᴍ, the incubation with pyridoxal phosphate reconstituted the active complex (holoenzyme) following a second order kinetic with a k
of 5.2 min
mᴍ
. This active complex showed a dissociation constant (K
) of 7.8 x 10
ᴍ. For concentrations higher than 0.1 mᴍ, 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 mᴍ lysine also a partial reactivation (among 20-33%) is observed. The enzyme treated with 1 mᴍ pyridoxal phosphate has a different behavior in Sepharose chromatography indicating that the modified enzyme presents a smaller size due to a conformational change.