Interactions between apoaspartate aminotransferase and pyridoxal 5'-phosphate. A stopped-flow study.

The fast kinetics and mechanism of the reconstitution reaction of holoaspartate aminotransferase from apoenzyme and pyridoxal 5'-phosphate were investigated by the stopped-flow method. When the absorbance change was monitored at 362 nm, the process was shown to involve three steps. The dependence of the three relaxation times on pyridoxal 5'-phosphate concentration and the analysis of the amplitudes enabled us to propose a mechanism in which the initial reversible binding step was followed by two irreversible isomerization steps. The rate constants and the extinction coefficients at 362 nm of the intermediate species were determined. Studies of the reconstitution under the stoichiometric conditions at various wavelengths confirmed the occurrence of at least three steps, and especially of the last decoupled step, but strongly suggest that the actual mechanism is more complex.