title: A Secondary beta Deuterium Kinetic Isotope Effect in the Chorismate Synthase Reaction.
abstract: Chorismate synthase (EC 4.6.1.4) is the shikimate pathway enzyme that catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The enzyme reaction is unusual because it involves a trans-1,4 elimination of the C-3 phosphate and the C-6 proR hydrogen and it has an absolute requirement for reduced flavin. Several mechanisms have been proposed to account for the cofactor requirement and stereochemistry of the reaction, including a radical mechanism. This paper describes the synthesis of [4-(2)H]EPSP and the observation of kinetic isotope effects using this substrate with both Neurospora crassa and Escherichia coli chorismate synthases. The magnitude of the effects were (D)(V) = 1.08 +/- 0.01 for the N. crassa enzyme and 1.10 +/- 0.02 on phosphate release under single-turnover conditions for the E. coli enzyme. The effects are best rationalised as substantial secondary beta isotope effects. It is most likely that the C(3)-O bond is cleaved first in a nonconcerted E1 or radical reaction mechanism. Although this study alone cannot rule out a concerted E2-type mechanism, the C(3)-O bond would have to be substantially more broken than the proR C(6)-H bond in a transition state of such a mechanism. Importantly, although the E. coli and N. crassa enzymes have different rate limiting steps, their catalytic mechanisms are most likely to be chemically identical. Copyright 2000 Academic Press.
authors.affiliation: Biological Chemistry Department, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom
authors.initials: S
authors.lastname: Bornemann
authors.initials: ME
authors.lastname: Theoclitou
authors.initials: M
authors.lastname: Brune
authors.initials: MR
authors.lastname: Webb
authors.initials: RN
authors.lastname: Thorneley
authors.initials: C
authors.lastname: Abell