Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution

Journal Article · Tue Apr 17 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00467a011· OSTI ID:6274326

Schmidheini, T; Moesch, H U; Braus, G ^[1]; Evans, J N.S. ^[2]

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1. Eidgenoessische Technische Hochschule, Zuerich (Switzerland)
2. Washington State Univ., Pullman (USA)

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 M{sub r} 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 {sup 1}H 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){sub 0.5} value of 4.0 mM. In the presence of the activator tryptophan, the cooperativity is lost. The enzyme has an (S){sub 0.5} value of 1.2 mM in the presence of 10 {mu}M tryptophan and an increased (S){sub 0.5} value of 8.6 mM in the presence of 300 {mu}M tyrosine. In the mutant enzyme, a loss of the cooperativity was observed, and (S){sub 0.5} was reduced to 1.0 mM. This enzyme is therefore locked in the activated state by a single amino acid substitution.

• OSTI ID: 6274326
• Journal Information: Biochemistry; (USA), Vol. 29:15; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

62 RADIOLOGY AND NUCLEAR MEDICINE
OXIDOREDUCTASES
NUCLEAR MAGNETIC RESONANCE
AMINO ACID SEQUENCE
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
MUTANTS
PROTONS
PURIFICATION
SACCHAROMYCES CEREVISIAE
TRYPTOPHAN
TYROSINE
AMINO ACIDS
AROMATICS
AZAARENES
AZOLES
BARYONS
CARBOXYLIC ACIDS
ELEMENTARY PARTICLES
ENZYMES
EUMYCOTA
FERMIONS
FUNGI
HADRONS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROXY ACIDS
INDOLES
KINETICS
LABELLED COMPOUNDS
MAGNETIC RESONANCE
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PLANTS
PYRROLES
REACTION KINETICS
RESONANCE
SACCHAROMYCES
YEASTS
550601* - Medicine- Unsealed Radionuclides in Diagnostics