KINETIC CHARACTERIZATION OF CHANNEL IMPAIRED MUTANTS OF TRYPTOPHAN SYNTHASE

Tryptophan synthase, an alpha(2) beta(2) tetrameric complex, is a clas sic example of an enzyme that is thought to ''channel'' a metabolic in termediate (indole) from the active site of the alpha subunit to the a ctive site of the beta subunit. The solution of the three-dimensional structure of the enzyme from Salmonella typhimurium provided physical evidence for a 25-Angstrom hydrophobic tunnel which connects the alpha and beta active sites (Hyde, C. C., Ahmed, S. A, Padlan, E. A., Miles , E. W., and Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871). Us ing rapid reaction kinetics, we have previously established that indol e is indeed channeled and have identified three essential kinetic feat ures which govern efficient channeling. In the current study we have p robed the necessity of these features by using site-directed mutagenes is to alter these requirements. We now report the kinetic characteriza tion of two mutants which contain substitutions to block or restrict t he tunnel (beta C170F and beta C170W). Preliminary kinetic and structu ral evidence of a restricted tunnel in the beta C170W has been provide d (Schlichting, I., Yang, X W., Miles, E.W., Kim, A. Y., and Anderson, K. S. (1994) J. Biol. Chem. 269, 26591-26593). The rapid kinetic anal ysis of these mutant proteins shows that these mutations interfere wit h efficient channeling of the indole metabolite such that indole can b e observed in single enzyme turnover of the physiologically relevant a lpha beta reaction. In addition, the beta C170W mutant appears to be i mpaired in alpha beta intersubunit communication.