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

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Abstract

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 Nα‐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.

Original languageEnglish
Pages (from-to)531-538
Number of pages8
JournalEuropean Journal of Biochemistry
Volume133
Issue number3
DOIs
StatePublished - Jul 1983

ASJC Scopus subject areas

  • Biochemistry

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