Cellular enzyme-substrate complexes: Probes for mass spectrometric detection
- Published: Apr 15, 2017
- Author: Steve Down
- Channels: Base Peak
Cellular enzyme reactions
Living cells are home to a host of reactions but our knowledge of them is fairly limited. Although we are familiar with the existence of many cellular enzymes, the exact functions of a high proportion remain unclear and the substrates that they react with unknown. In some processes, we know which substrate reacts with which enzyme but the exact structure of the enzyme is ill-defined, with a number of forms, produced by post-translational modifications and other variations, in the frame.
The complex medium that is the cellular milieu has attracted the particular attention of scientists in the US, who have devised a new platform for identifying enzyme-substrate pairs. Vicki Wysocki and Jing Yan from Ohio State University and Zhaohui Sunny Zhou, Kalli Catcott and Wanlu Qu from Northeastern University acknowledged that several screening techniques already exist for matching a substrate and an enzyme but they have proved to be inadequate in cells because they cannot distinguish between specific enzyme-substrate pairs.
Their solution has been dubbed isotope-labelled, activity-based identification and tracking, which collapses into the handy acronym IsoLAIT. It relies on the combination of a special probe that binds both to the enzyme and the substrate to create a trimodal complex that remains intact when it is subjected to mass spectrometric analysis. The method is strengthened by introducing isotope labels into the probe so that the complex can be identified unambiguously without any prior knowledge of the structures of the three components.
The new protocol was demonstrated with an enzyme called thiopurine methyltransferase (TPMT) which functions in cells by adding a methyl group to the thiol function in thiophenols. From previous studies, the researchers knew that TPMT catalysed the formation of a complex between the thiol group of a thiophenol and the probe S-adenosyl-L-vinthionine (AdoVin). However, they did not know if the enzyme-substrate-probe complex could survive passage through the mass spectrometer and be detected intact.
As a test case, 2-nitro-5-thiobenzoic acid was incubated with TPMT and a mixture of natural and isotope-labelled AdoVin. After transfer to a suitable buffer, the solution was added to a glass capillary for infusion into a hybrid ion mobility-time-of-flight mass spectrometer fitted with a nano-electrospray ionisation source. The minimal sample preparation and gentle ionisation conditions are conducive to maintaining the three components together in the complex.
Examination of the mass spectra clearly showed multiple-charged peaks corresponding to the intact complex as well as the free enzyme and the two entities were clearly distinguished from each other. Under collision-induced dissociation, the trimodal complex broke down into the free enzyme and the substrate-probe complex. The latter was clearly identified from the doublet signals originating from the unlabelled and isotope-labelled probes which were 15 Da apart.
So, the procedure worked for in vitro samples but would it be as successful for cellular systems? Using the supernatant from TPMT-transformed E. coli cells with 2-nitro-5-thiobenzoic acid and AdoVin, the three-component complex was again observed by mass spectrometry. In this case, the complex was not fully resolved in the spectrum from the free enzyme, possibly due to peak overlap, but its presence was confirmed unequivocally.
General strategy for enzymes
One interesting find was that the mass-to-charge ratios of the substrate-probe complexes from the in vitro and ex vivo samples were different, meaning that some sort of modification had taken place. In fact, the nitro group of 2-nitro-5-thiobenzoic acid in the cellular sample had been reduced to the amine, probably by a nitroreductase that was also present in the cells. So, the amine-containing substrate-probe adduct was observed rather than the nitro one.
The research team suggested that this small but significant modification in the structure might not have been picked up by conventional methods. However, the IsoLAIT procedure looks for mass patterns originating from the probe, rather than specific structures, so the change was easily found.
The power of IsoLAIT was demonstrated by the failure to identify the enzyme-substrate complex in the absence of the probe, due to its inherent instability under the mass spectrometer conditions. The method could also be improved by introducing sample purification and chromatographic separation before mass spectrometric analysis as long as these processes maintained the integrity of the enzyme-substrate-probe complex.
This is the first reported observation of cellular enzyme-substrate complexes by mass spectrometry. The method can be adapted using different probes so that other enzyme-substrate pairs can be IsoLAITed and has the potential to become a general approach for identifying substrates for particular enzymes within cells.
ChemBioChem 2017, 18, 613-617: "Identifying Unknown Enzyme–Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry"
Article by Steve Down
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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