A secondary beta deuterium kinetic isotope effect in the chorismate synthase reaction

Chorismate synthase (EC 4.6.1.4) is the shikimate pathway enzyme that catal yzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorism ate. The enzyme reaction is unusual because it involves a trans-1,4 elimina tion 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 ac count for the cofactor requirement and stereochemistry of the reaction, inc luding a radical mechanism. This paper describes the synthesis of [4-H-2]EP SP and the observation of kinetic isotope effects using this substrate with both Neurospora crassa and Escherichia coli chorismate synthases. The magn itude of the effects were (D)(V) = 1.08 +/- 0.01 for the N. crassa enzyme a nd 1.10 +/- 0.02 on phosphate release under single-turnover conditions for the E. coli enzyme. The effects are best rationalised as substantial second ary 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 st udy alone cannot rule out a concerted E2-type mechanism, the C(3)-O bond wo uld have to be substantially more broken than the proR C(6)-H bond in a tra nsition state of such a mechanism. Importantly, although the E. coli and N. crassa enzymes have different rate limiting steps, their catalytic mechani sms are most likely to be chemically identical. (C) 2000 Academic Press.