Detection and identification of transient enzyme intermediates using rapid mixing, pulsed-flow electrospray mass spectrometry

Rapid chemical quench methods coupled with off-line detection have proven to be very useful in identifying enzyme reaction intermediates, However: a limitation to this approach involves enzyme intermediates which are too labile under the chemical quenching conditions to allow detection and characterization, In this report, we describe the development of a novel approach for the detection and characterization of enzyme intermediates on the subsecond time scale using a ''pulsed flow'' method which employs a direct Interface between a rapid-mixing device and electrospray ionization mass spectrometry. The application of this technique with the enzyme 5-enolpyruvoyl-shikimate-3-phosphate (EPSP) synthase is demonstrated, This enzyme converts shikimate-3-phosphate (S3P) and phosphoenol pyruvate (PEP) to EPSP and inorganic phosphate. Previous rapid chemical quench studies have shown that this reaction proceeds through a tetrahedral intermediate [Anderson, K. S., rt al. (1988) J. Am. Chem. Soc. 110, 6577-6579] formed transiently at the enzyme active site, We have shown that this tetrahedral intermediate can be directly detected on a subsecond time scale 1 without chemical quenching by interfacing a rapid mixing apparatus directly with an on-line electrospray ionization ion trap mass spectrometer. Negative ion mass spectra collected by electrospray ionization indicate peaks for S3P (m/z 253), PEP(m/z 167), EPSP (m/z 323), and the tetrahedral intermediate (m/z 421). Further confirmation was provided by performing the same experiment-with [C-13-1]-labeled PEP, These spectra confirmed the anticipated shift of 1 atomic mass unit for PEP (m/z 168), EPSP (m/z 324), and the tetrahedral intermediate (m/z 422) with no change in S3P (m/z 253). Thr collision-induced dissociation of the unlabeled tetrahedral intermediate peak (m/z 421) produced a daughter ion at m/z 323, which is most likely EPSP resulting from the loss of phosphate and is consistent with previous studies which have examined the chemical breakdown of the tetrahedral intermediate In solution [Anderson, K. S., et al. (1990) J. Biol. Chem. 265, 5567-6672], This technique is under development and should be a useful method to study the transient formation of enzyme intermediates.