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 pro ven to be very useful in identifying enzyme reaction intermediates, Ho wever: a limitation to this approach involves enzyme intermediates whi ch are too labile under the chemical quenching conditions to allow det ection and characterization, In this report, we describe the developme nt of a novel approach for the detection and characterization of enzym e intermediates on the subsecond time scale using a ''pulsed flow'' me thod which employs a direct Interface between a rapid-mixing device an d electrospray ionization mass spectrometry. The application of this t echnique with the enzyme 5-enolpyruvoyl-shikimate-3-phosphate (EPSP) s ynthase is demonstrated, This enzyme converts shikimate-3-phosphate (S 3P) and phosphoenol pyruvate (PEP) to EPSP and inorganic phosphate. Pr evious rapid chemical quench studies have shown that this reaction pro ceeds through a tetrahedral intermediate [Anderson, K. S., rt al. (198 8) J. Am. Chem. Soc. 110, 6577-6579] formed transiently at the enzyme active site, We have shown that this tetrahedral intermediate can be d irectly detected on a subsecond time scale 1 without chemical quenchin g by interfacing a rapid mixing apparatus directly with an on-line ele ctrospray ionization ion trap mass spectrometer. Negative ion mass spe ctra 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 exp eriment-with [C-13-1]-labeled PEP, These spectra confirmed the anticip ated shift of 1 atomic mass unit for PEP (m/z 168), EPSP (m/z 324), an d the tetrahedral intermediate (m/z 422) with no change in S3P (m/z 25 3). Thr collision-induced dissociation of the unlabeled tetrahedral in termediate 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 consiste nt with previous studies which have examined the chemical breakdown of the tetrahedral intermediate In solution [Anderson, K. S., et al. (19 90) J. Biol. Chem. 265, 5567-6672], This technique is under developmen t and should be a useful method to study the transient formation of en zyme intermediates.