Time-Resolved Solid-State NMR Spectroscopy of 5-Enolpyruvylshikimate-3-phosphate Synthase

Abstract

The novel technique of time-resolved solid-state NMR spectroscopy has been used to characterize the enzyme, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, in both the forward and reverse directions over time periods ranging from 5 to 300 ms. The wealth of data currently available for EPSP synthase, in particular the pre-steady-state kinetics performed using chemical quench-flow experiments [Anderson, K. S., Sikorski, J. A., & Johnson, K. A. (1988) Biochemistry 27, 7395-7406], has made the enzyme an obvious choice as a proving ground for this new technique. Pre-steady-state 13C TOSS CP-MAS spectra have been obtained with a much improved signal-to-noise ratio, and corrections have been made to some previously reported assignments [Evans, J.N.S., Appleyard, R.J., & Shuttleworth, W.A. (1993) J. Am. Chem. Soc. 115, 1588-1590]. Peak fitting has allowed the extrapolation of NMR integral intensities of species involved in the reaction. These show a good correlation with concentrations calculated by simulations using the kinetic parameters obtained from the chemical quench-flow experiments. It is proposed that careful optimization of the contact time used will be necessary to obtain accurate, relative concentrations that will enable an independent kinetic simulation by time-resolved solid-state NMR. The technique shows much promise due to its nondestructive quenching procedure, which allows the direct observation of enzyme intermediates on a reaction pathway. However, its requirement of significantly larger amounts of enzyme does limit the technique to those proteins which naturally occur in high abundance or have been hyperexpressed.