The Accessibility of the Active Site and Conformation States of the β₂ Subunit of Tryptophan Synthase Studied by Fluorescence Quenching

The rate of quenching of the fluorescence of pyridoxal 5′‐phosphate in the active site of the β₂ subunit of tryptophan synthase from Escherichia coli was measured to estimate the accessibility of the coenzyme to the small molecules iodide and acrylamide. The α subunit and the substrate l‐serine substantially reduced the quenching rate. For iodide, the order of decreasing quenching was: Schiff's base of N^α‐acetyl‐lysine with pyridoxal 5′‐phosphate > holoβ₂ subunit > holoα₂β₂ complex ∼ holoβ₂ subunit +l‐serine > holoα₂β₂ complex +l‐serine. The coenzyme in the β₂ subunit is apparently freely accessible to both iodide and acrylamide (k∼× 10⁹ M⁻¹ s⁻¹), but the α subunit and l‐serine decrease the rate by factors of 2–5. Quenching of the fluorescence of the single tryptophan residue of the β₂ subunit revealed that the apo and holo forms exist in different states, whereas the α subunit stabilises a third conformation. As the α subunit binds to the β₂ subunit, the tryptophan residue, which is within 2.2 nm of the active site of the β₂ subunit, probably rotates with respect to the plane of the ring of the coenzyme, such that fluorescence energy transfer from tryptophan to pyridoxal phosphate is greatly reduced. The α subunit strongly protects the active‐site ligand indole propanol phosphate from quenching with acrylamide, consistent with the active site being deep in a cleft in the protein. Iodide induces dissociation of the holoα₂β₂ complex [E. W. Miles & M. Moriguchi (1977) J. Biol. Chem. 252, 6594–6599]. The effect of iodide on the fluorescence properties of holoα₂β₂ complex allows us to estimate an upper limit for the dissociation constant for the α₂β₂ complex of 10^‐8M, in the absence of iodide.