Mechanism of reconstitution of the apo.beta.2 subunit and the .alpha.2apo.beta.2 complex of tryptophan synthase with pyridoxal 5'-phosphate: kinetic studies

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Abstract
The mechanism of pyridoxal 5''-phosphate (PLP) binding to the .alpha.2apo.beta.2 complex and the apo.beta.2 subunit of [Escherichia coli] 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.2apo.beta.2 complex binds PLP in a sequence of 3 steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by formation of an active .alpha.2holo.beta.2 complex. The 2 binding sites appear to bind PLP independently. The apo.beta.2 subunit binds PLP cooperatively in a sequence of 3 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 5''-phosphate (PNP) binding, the rate data of the apo.beta.2 subunit are consistent with the concerted mechanism. The differences 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.