Mechanism of reconstitution of the apo beta 2 subunit and the alpha 2 apo beta 2 complex of tryptophan synthase with pyridoxal 5'-Phosphate: kinetic studies.
The mechanism of pryidoxal 5'-phosphate (PLP) binding to both the alpha apo beta 2 complex and the apo beta 2 subunit of tryptophan synthase was investigated by rapid mixing experiments. Absorption and fluorescence changes were used to monitor the binding reaction directly. Reduction with sodium borohydride provided the rate of formation of the internal aldimine with the lysine amino group of the enzyme, and substrate turnover monitored the rate of formation of active enzyme. The alpha 2 apo beta 2 complex binds PLP in a sequence of three steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by formation of an active alpha 2 holo beta 2 complex. The two binding sites appear to bind PLP independently. The apo beta 2 subunit binds PLP cooperatively in a sequence of three steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by the formation of the enzymically active holo beta 2 subunit. Taken together with kinetic studies of pyridoxine phosphate binding [Tschopp, J., & Kirschner, K. (1980) Biochemistry (second paper of three in this issue)], the rate data of the apo beta 2 subunit are shown to be consistent with the concerted mechanism. The difference between the values of the isomerization rate constants of bound PLP and bound PNP appear to result from the covalent internal aldimine, which is formed with PLP but not with PNP.
D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D014367:Tryptophan Synthase / Q000378:metabolism*
D001051:Apoenzymes; D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D014367:Tryptophan Synthase