The Accessibility of the Active Site and Conformation States of the β 2 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 β 2 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 > holoβ 2 subunit > holoα 2 β 2 complex ∼ holoβ 2 subunit + l ‐serine > holoα 2 β 2 complex + l ‐serine. The coenzyme in the β 2 subunit is apparently freely accessible to both iodide and acrylamide ( k ∼× 10 9 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 β 2 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 β 2 subunit, the tryptophan residue, which is within 2.2 nm of the active site of the β 2 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 holoα 2 β 2 complex [E. W. Miles & M. Moriguchi (1977) J. Biol. Chem. 252 , 6594–6599]. The effect of iodide on the fluorescence properties of holoα 2 β 2 complex allows us to estimate an upper limit for the dissociation constant for the α 2 β 2 complex of 10 ‐8 M, in the absence of iodide.