ESCHERICHIA-COLI CHORISMATE SYNTHASE - A DEUTERIUM KINETIC-ISOTOPE EFFECT UNDER SINGLE-TURNOVER AND STEADY-STATE CONDITIONS SHOWS THAT A FLAVIN INTERMEDIATE FORMS BEFORE THE C-(6PROR)-H BOND IS CLEAVED

We report the observation of a deuterium kinetic isotope effect for th e conversion of 5-enolpyruvylshikimate-3-phosphate into chorismate (V- 6proR2H=1.13+/-0.03) using recombinant chorismate mate synthase from E scherichia coli. Similar isotope effects were observed for the decay o f a spectroscopically characterized flavin intermediate ((6proR2H)k = 1.17+/-0.04) during single-turnover experiments. The main rate-limitin g steps and C-(6proR)-H bond breaking are therefore distinct and both must occur after the formation of the flavin intermediate and either b efore or concomitant with its decay.