Paiva A A, Tilton R F, Crooks G P, Huang L Q, Anderson K S
Bayer Corporation, Pharmaceutical Division, 400 Morgan Lane, West Haven, Connecticut 06516, USA.
Biochemistry. 1997 Dec 9;36(49):15472-6. doi: 10.1021/bi971883i.
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.
快速化学淬灭方法与离线检测相结合已被证明在鉴定酶反应中间体方面非常有用。然而，这种方法的一个局限性涉及到一些酶中间体，它们在化学淬灭条件下过于不稳定，无法进行检测和表征。在本报告中，我们描述了一种使用“脉冲流”方法在亚秒时间尺度上检测和表征酶中间体的新方法，该方法采用了快速混合装置与电喷雾电离质谱之间的直接接口。展示了该技术在5-烯醇丙酮酸莽草酸-3-磷酸（EPSP）合酶上的应用。这种酶将莽草酸-3-磷酸（S3P）和磷酸烯醇丙酮酸（PEP）转化为EPSP和无机磷酸。先前的快速化学淬灭研究表明，该反应通过在酶活性位点瞬时形成的四面体中间体进行[安德森，K.S.等人（1988年）《美国化学会志》110，6577 - 6579]。我们已经表明，通过将快速混合装置直接与在线电喷雾电离离子阱质谱仪连接，可以在不进行化学淬灭的情况下在亚秒时间尺度上直接检测到这种四面体中间体。通过电喷雾电离收集的负离子质谱显示了S3P（m/z 253）、PEP（m/z 167）、EPSP（m/z 323）和四面体中间体（m/z 421）的峰。用[13C - 1]标记的PEP进行相同实验提供了进一步的证实。这些质谱证实了PEP（m/z 168）、EPSP（m/z 324）和四面体中间体（m/z 422）预期的1个原子质量单位的位移，而S3P（m/z 253）没有变化。未标记的四面体中间体峰（m/z 421）的碰撞诱导解离产生了一个m/z 323的子离子，这很可能是由于磷酸丢失导致的EPSP，并且与先前研究溶液中四面体中间体化学分解的研究一致[安德森，K.S.等人（1990年）《生物化学杂志》265，5567 - 6672]。该技术正在开发中，应该是研究酶中间体瞬时形成的一种有用方法。
