The kinetics of Schiff-base formation during reconstitution of D-serine apodehydratase from Escherichia coli with pyridoxal 5'-phosphate.
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 x 10(6) 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.
D001051:Apoenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D012695:L-Serine Dehydratase / Q000378:metabolism*; D008239:Lysine; D008623:Mercaptoethanol; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D012545:Schiff Bases; D013057:Spectrum Analysis; D013696:Temperature
D001051:Apoenzymes; D012545:Schiff Bases; D011732:Pyridoxal Phosphate; D008623:Mercaptoethanol; D012695:L-Serine Dehydratase; D008239:Lysine