title: Evidence that cysteine 298 is in the active site of tryptophan indole-lyase.
abstract: Escherichia coli tryptophan indole-lyase (tryptophanase) mutants, with cysteine residues 294 and 298 selectively replaced by serines, have been prepared by site-directed mutagenesis. Both mutant enzymes are highly active for beta-elimination reactions measured with both L-tryptophan and S-(o-nitrophenyl)-L-cysteine. The Cys-294----Ser mutant enzyme is virtually identical to the wild type with respect to pyridoxal phosphate binding (KCO = 2 microM), cofactor absorption spectrum (lambda max = 420 and 337 nm) and pH dependence (pK alpha = 7.3), pH profile for catalysis, and rate of bromopyruvic acid inactivation. In contrast, the Cys-298----Ser mutant enzyme exhibits a reduced affinity for pyridoxal phosphate (KCO = 6 microM), a shift in the cofactor absorption spectrum to 414 nm and an altered pK alpha = 8.5, an alkaline shift in the pH profile for catalysis, and resistance to inactivation of the apoenzyme by bromopyruvic acid. The C298S mutant enzyme (wherein cysteine 298 is altered to serine) also undergoes an isomerization to an unreactive state upon storage at 4 degrees C. These results demonstrate that the sulfhydryl groups of Cys-294 and Cys-298 are catalytically nonessential. However, these data suggest that Cys-298 is located within or very near the active site of the enzyme and is the reactive cysteine residue previously observed by others.
authors.affiliation: Department of Chemistry, School of Chemical Sciences, University of Georgia, Athens 30602.
authors.forename: R S
authors.initials: RS
authors.lastname: Phillips
authors.forename: P D
authors.initials: PD
authors.lastname: Gollnick
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D005796:Genes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D014368:Tryptophanase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D008190:Lyases; D014368:Tryptophanase; D003545:Cysteine