Equilibrium and Kinetic Measurements of the Binding of Pyridoxal 5′‐Phosphate to Hybrid Tryptophan Synthase from Escherichia coli

Hybrid tryptophan synthase was prepared that contains a β, subunit with one functional active site while the internal aldimine between cofactor and enzyme of the other protomer was reduced with sodium borohydride (α,ββ*). The modified enzyme shows a specific activity for the l ‐serine+indole to l ‐tryptophan reaction of about 50% compared to the native enzyme [Hathaway, G. M., Kida, S. and Crawford, I. P., Biochemistry 8 , 989—997 (1969)]. The binding of pyridoxal 5′‐phosphate (pyridoxal‐ P ) to the α 2 apo ββ* complex and to the apo ββ* subunit was studied by equilibrium methods and rapid mixing experiments. The equilibrium binding curves for the hybrid species are hyperbolic (i. e. non‐cooperative), yielding apparent microscopic dissociation constants of 1.6±O.2 × 10 −6 M for the α 2 holoββ* subunit. The rate of formation of the internal aldimine in the unmodified active site was followed by fast reduction with sodium borohydride, L‐tryptophan synthesis was used to monitor the rate of formation of active enzyme. Cofactor binding to the α 2 apo ββ* complex is characterized by three sequential steps of decreasing rate: formation of a non‐covalent initial complex, which reacts to an enzymatically inactive internal aldimine, followed by a conformational change leading to the active α 2 holo ββ* bienzyme complex. A similar series of consecutive steps with decreasing reaction rates is observed for the binding of pyridoxal‐ P to the apoββ* subunit. The formation of active enzyme, however, is biphasic. The faster process, which parallels the formation of the internal aldimine and which accounts for 85% of the total amplitude, obviously involves an already active high‐affinity state of the hybrid molecule whereas the second phase parallels the activation process as observed for cofactor binding to the native dimer. These findings are discussed with respect to the recently proposed mechanism for pyridoxal‐ P binding to the unmodified apoβ 2 subunit [Bartholmes, P. Balk, H. and Kirschner, K, Biochemistry 19 , 4527–4533 (1980]