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., et 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 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/z323), and the tetrahedral intermediate (m/z 421). Further confirmation was provided by performing the same experiment with [13C-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). The collision-induced dissociation of the unlabeled tetrahedral intermediate peak (m/z421) 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.
Paiva
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Bayer Corporation, Pharmaceutical Division, 400 Morgan Lane, West Haven, Connecticut 06516, USA.
Tilton
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RF
Crooks
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Huang
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LQ
Anderson
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KS
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000737:chemistry*; D002384:Catalysis; D013058:Mass Spectrometry / Q000379:methods*
D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase