The catalytic mechanism of EPSP synthase revisited

Recent analysis of EPSP synthase by solid-state NMR has led to the postulation of a new enzyme reaction pathway and raised once again the question of an intermediate species covalently bound to the enzyme [Studelska, D., McDowell, L., Espe, M., Klug, C., and Schaefer, J. (1997) Biochemistry 36, 15555-15560]. Therefore, we have reexamined the mechanism of the reaction catalyzed by EPSP synthase and analyzed the reaction products formed under the conditions used in preparing samples for solid-state NMR. Single-turnover experiments were carried out using both [1-C-14]- and [P-32]PEP showing the formation and decay of the previously proposed tetrahedral intermediate species on a time scale comparable with the disappearance of substrate and formation of product, thus unequivocally establishing the kinetic competence. The possible presence of a covalently bound enzyme intermediate species was also investigated, using SDS-PAGE and Centricon concentration analysis of the quenched reaction samples. No covalently bound enzyme intermediates were observed during the reaction. An enzyme assay was also performed repeating the conditions used in sample preparation for the solid-state NMR studies. We show that under these conditions, total turnover of substrates to products was observed within 45 s at -30 degrees C prior to freezing and lyophilization, Following lyophilization, the samples were stored at -20 degrees C and analyzed over a period of 21 days. We observed the conversion of the product EPSP into the side product, a cyclic EPSP ketal, and the breakdown product, pyruvate, Thus, the new species reported by solid-state NMR can be accounted for by previously characterized reaction products and side products formed during sample preparation and upon incubation in the solid-state. Our conclusions are also supported by the solution and solid-state NMR studies recently reported [Jakeman et al. (1998) Biochemistry 37, 12012-12019]. These results once again highlight the importance of kinetic competence as a criterion to be used in defining enzyme intermediates and point to the errors in interpretation of results when the time dependence of formation of the proposed intermediates is not considered.