Mechanism of the physiological reaction catalyzed by tryptophan synthase from Escherichia coli.
The physiological synthesis of L-tryptophan from indoleglycerol phosphate and L-serine catalyzed by the alpha 2 beta 2 bienzyme complex of tryptophan synthase requires spatial and dynamic cooperation between the two distant alpha and beta active sites. The carbanion of the adduct of L-tryptophan to pyridoxal phosphate accumulated during the steady state of the catalyzed reaction. Moreover, it was formed transiently and without a lag in single turnovers, and glyceraldehyde 3-phosphate was released only after formation of the carbanion. These and further data prove first that the affinity for indoleglycerol phosphate and its cleavage to indole in the alpha subunit are enhanced substantially by aminoacrylate bound to the beta subunit. This indirect activation explains why the turnover number of the physiological reaction is larger than that of the indoleglycerol phosphate cleavage reaction. Second, reprotonation of nascent tryptophan carbanion is rate limiting for overall tryptophan synthesis. Third, most of the indole generated in the active site of the alpha subunit is transferred directly to the active site of the beta subunit and only insignificant amounts pass through the solvent. Comparison of the single turnover rate constants with the known elementary rate constants of the partial reactions catalyzed by the alpha and beta active sites suggests that the cleavage reaction rather than the transfer of indole or its condensation with aminoacrylate is rate limiting for the formation of nascent tryptophan.
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Abteilung Biophysikalische Chemie, Biozentrum der Universität Basel, Switzerland.
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D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013057:Spectrum Analysis; D014364:Tryptophan / Q000096:biosynthesis; D014367:Tryptophan Synthase / Q000378:metabolism*
D007211:Indoles; D009097:Multienzyme Complexes; D005986:Glyceraldehyde 3-Phosphate; D012694:Serine; D014364:Tryptophan; D014367:Tryptophan Synthase