Kinetic characterization of channel impaired mutants of tryptophan synthase.
Tryptophan synthase, an alpha 2 beta 2 tetrameric complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. The solution of the three-dimensional structure of the enzyme from Salmonella typhimurium provided physical evidence for a 25-A 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). Using rapid reaction kinetics, we have previously established that indole is indeed channeled and have identified three essential kinetic features which govern efficient channeling. In the current study we have probed the necessity of these features by using site-directed mutagenesis to alter these requirements. We now report the kinetic characterization of two mutants which contain substitutions to block or restrict the tunnel (beta C170F and beta C170W). Preliminary kinetic and structural evidence of a restricted tunnel in the beta C170W has been provided (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 analysis of these mutant proteins shows that these mutations interfere with efficient channeling of the indole metabolite such that indole can be observed in single enzyme turnover of the physiologically relevant alpha beta reaction. In addition, the beta C170W mutant appears to be impaired in alpha beta intersubunit communication.
Anderson
K S
KS
Yale University School of Medicine, Department of Pharmacology, New Haven, Connecticut 06520, USA.
Kim
A Y
AY
Quillen
J M
JM
Sayers
E
E
Yang
X J
XJ
Miles
E W
EW
D001665:Binding Sites; D002250:Carbon Radioisotopes; D005994:Glycerophosphates / Q000378:metabolism*; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008962:Models, Theoretical; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011865:Radioisotope Dilution Technique; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*; D012694:Serine; D013329:Structure-Activity Relationship; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002250:Carbon Radioisotopes; D005994:Glycerophosphates; D007211:Indoles; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C008117:indoleglycerol phosphate; D012694:Serine; C030374:indole; D014367:Tryptophan Synthase