TIME-RESOLVED SOLID-STATE NMR-SPECTROSCOPY OF 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE

The novel technique of time-resolved solid-state NMR spectroscopy has been used to characterize the enzyme, 5-enolpyruvylshikimate-3-phospha te (EPSP) synthase, in both the forward and reverse directions over ti me periods ranging from 5 to 300 ms. The wealth of data currently avai lable for EPSP synthase, in particular the pre-steady-state kinetics p erformed using chemical quench-flow experiments [Anderson, K. S., Siko rski, J. A., & Johnson, K. A. (1988) Biochemistry 27, 7395-7406], has made the enzyme an obvious choice as a proving ground for this new tec hnique. Pre-steady-state C-13 TOSS CP-MAS spectra have been obtained w ith a much improved signal-to-noise ratio, and corrections have been m ade to some previously reported assignments [Evans, J. N. S.; Appleyar d, R. J., & Shuttleworth, W. A. (1993) J. Am. Chem. Sec. 115, 1588-159 0]. Peak fitting has allowed the extrapolation of NMR integral intensi ties of species involved in the reaction. These show a good correlatio n with concentrations calculated by simulations using the kinetic para meters obtained from the chemical quench-flow experiments. It is propo sed that careful optimization of the contact time used will be necessa ry to obtain accurate, relative concentrations that will enable an ind ependent kinetic simulation by time-resolved solid-state NMR. The tech nique shows much promise due to its nondestructive quenching procedure , which allows the direct observation of enzyme intermediates on a rea ction pathway. However, its requirement of significantly larger amount s of enzyme does limit the technique to those proteins which naturally occur in high abundance or have been hyperexpressed.