The Quarternary Structure of Tryptophan Synthase from Escherichia coli Fluorescence and Hydrodynamic Studies

Andrew N. LANE, Kasper KIRSCHNER

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19 Scopus citations


Single cysteine residues on the separated α and β2 subunits of tryptophan synthase were labelled with various fluorescent ligands suitable for fluorescence energy transfer measurements. Labelled α subunits, reconstituted into an αL2β2 complex, retained more than 75% activity. Labelled β2 subunits, either free or reconstituted into an α2βL2′ complex, retained 10–30% of their activity, depending on the label. The coenzyme pyridoxal 5′‐phosphate served as a natural fluorescent label of the active site of the β2 subunit. Different combinations of labelled subunits resulted in doubly labelled αL2βL2′ complexes that allowed five independent distances to be estimated by fluorescence energy transfer: within one β protomer, CysL–pyridoxal 5′‐phosphate = 1.4–3.2 nm; between two β protomers, CysL–CysL= 0.5–2.3 nm; between one α protomer and a β protomer, CysL–pyridoxal 5′‐phosphate = 1.9–3.2 nm, CysL–CysL= 3.1–4.4 nm; between two α protomers, CysL–CysL= 3.1–3.9 nm. Translational frictional ratios obtained from measurements of sedimentation and diffusion constants and partial specific volumes provided independent information on the shapes of the different particles in solution. Both the α and the β protomer are known to consist of two globular domains of known molecular weight. These were assumed to be approximately spherical and used to construct models first of the α and β2 subunits and then of the α2β2 complex. The data on specific distances and shape factors, together with considerations of symmetry and compactness allow many possible spatial arrangements of the domains to be eliminated. The radii of gyration, maximum dimensions and hydrated volumes calculated for the best models of the α and β2 subunits and the α2β2 complex agree reasonably well with the results of independent small‐angle X‐ray scattering studies [Wilhelm et al. (1982) Eur. J. Biochem. 129, 51–56. The active sites responsible for cleavage of indoleglycerol phosphate to indole and for condensation of indole with L‐serine are approximately 2 nm apart, ruling out the composite active sites hypothesis.

Original languageEnglish
Pages (from-to)675-684
Number of pages10
JournalEuropean Journal of Biochemistry
Issue number3
StatePublished - Jan 1983

ASJC Scopus subject areas

  • Biochemistry


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