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Mass spectrometric real-time monitoring of enzymatic glycosidic hydrolysis, enzymatic inhibition and enzyme complexes

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Abstract

The mass spectrometric development of an enzymatic assay resulting in enzymatic activity, its reaction pathway and its dissociation constant are described for the first time within a single experiment. The method combines the performance of a mass spectrometry-compatible enzyme assay with the direct detection of specific enzyme complexes using hen egg white lysozyme as a model. The continuous liquid-flow technique applied, when hyphenated with electrospray ionization (ESI)–time-of-flight (ToF)–mass spectrometry (MS), permitted the simultaneous detection of several substances involved in product screening as well as the direct observation of dissociation constants. Dissociation constants for the product inhibitor N, N′, N″-triacetylchitotriose were calculated using a Scatchard plot (12×10−6 M) and the law of mass action (18–24×10−6 M), and these are in good agreement with constants obtained in earlier mass spectrometric (6–18×10−6 M) or spectroscopic (6–8×10−6 M) studies. Finally, the enzymatic hydrolysis of glycosidic substrate was monitored by ESI–ToF–MS in the presence of various inhibitors, thus leading to decreased activities in terms of their enzyme affinities. The associated inhibitor–enzyme complexes could be detected for up to lower micromolar K d values.

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Acknowledgements

This work was supported in part by a grant from the Vereinigung zur Förderung der Milchwissenschaftlichen Forschung an der TUM in Freising-Weihenstephan e.V. and from the Bund der Freunde der Technischen Universität München e. V. The authors gratefully acknowledge Tamo Fukamizo for calculating the plot showing the degradation of NAG6 by HEWL and very helpful discussions.

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  1. Biopolymer Chemistry, Analytical Research Group, Department for Basic Life Sciences, Technical University of Munich, Weihenstephaner Berg 3, 85354, Freising–Weihenstephan, Germany

    Nicole Dennhart & Thomas Letzel

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  1. Nicole Dennhart
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  2. Thomas Letzel
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Correspondence to Thomas Letzel.

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Dennhart, N., Letzel, T. Mass spectrometric real-time monitoring of enzymatic glycosidic hydrolysis, enzymatic inhibition and enzyme complexes. Anal Bioanal Chem 386, 689–698 (2006). https://doi.org/10.1007/s00216-006-0604-1

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  • DOI: https://doi.org/10.1007/s00216-006-0604-1

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