Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution.
Chorismate mutase, a branch-point enzyme in the aromatic amino acid pathway of Saccharomyces cerevisiae, and also a mutant chorismate mutase with a single amino acid substitution in the C-terminal part of the protein have been purified approximately 20-fold and 64-fold from overproducing strains, respectively. The wild-type enzyme is activated by tryptophan and subject to feedback inhibition by tyrosine, whereas the mutant enzyme does not respond to activation by tryptophan nor inhibition by tyrosine. Both enzymes are dimers consisting of two identical subunits of Mr 30,000, each one capable of binding one substrate and one activator molecule. Each subunit of the wild-type enzyme also binds one inhibitor molecule, whereas the mutant enzyme lost this ability. The enzyme reaction was observed by 1H NMR and shows a direct and irreversible conversion of chorismate to prephenate without the accumulation of any enzyme-free intermediates. The kinetic data of the wild-type chorismate mutase show positive cooperativity toward the substrate with a Hill coefficient of 1.71 and a [S]0.5 value of 4.0 mM. In the presence of the activator tryptophan, the cooperativity is lost. The enzyme has an [S]0.5 value of 1.2 mM in the presence of 10 microM tryptophan and an increased [S]0.5 value of 8.6 mM in the presence of 300 microM tyrosine. In the mutant enzyme, a loss of cooperativity was observed, and [S]0.5 was reduced to 1.0 mM. This enzyme is therefore locked in the activated state by a single amino acid substitution.
Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Zürich, Switzerland.
T
T
Schmidheini
H U
HU
Mösch
J N
JN
Evans
G
G
Braus
D000595:Amino Acid Sequence; D001665:Binding Sites; D002826:Chorismate Mutase / Q000235:genetics* / Q000302:isolation & purification; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000235:genetics*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D007535:Isomerases; D002826:Chorismate Mutase