✍ Scribed by Stephen Bornemann; Maria-Elena Theoclitou; Martin Brune; Martin R. Webb; Roger N.F. Thorneley; Chris Abell
No coin nor oath required. For personal study only.
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 ) ϭ 1.08 Ϯ 0.01 for the N. crassa enzyme and 1.10 Ϯ 0.02 on phosphate release under single-turnover conditions for the E. coli enzyme. The effects are best rationalised as substantial secondary  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.
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