The Kinetics of Schiff‐Base Formation during Reconstitution of D‐Serine Apodehydratase from Escherichia coli with Pyridoxal 5′‐Phosphate

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Abstract
Schiff base formation during reconstitution of D‐serine dehydratase (Escherichia coli) from its apoenzyme and pyridoxal 5′‐phosphate (pyridoxal‐P) has been studied by rapid kinetic techniques using absorbance changes at 436 nm. Three distinct reaction phases have been observed. The first is a very rapid change during which pyridoxal‐P is initially bound to the apoenzyme. This step has an equilibrium constant of 1500 M−1 and a forward reaction rate of the order of 2.6×106 M−1 s−1. The second phase shows a first‐order rate constant with a value dependent on pyridoxal‐P and corresponds to a first‐order step with a forward rate constant of 3.04 s−1 interacting with the initial equilibrium. The final phase is a slow first‐order reaction, the rate constant of which is approximately 0.01 s−1 and is independent of pyridoxal‐P concentration. The active pyridoxal species has been shown to be the free pyridoxal‐P as opposed to hemiacetal or hemimercaptal forms.