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

Dennhart, Nicole; Letzel, Thomas
November 2006
Analytical & Bioanalytical Chemistry;Nov2006, Vol. 386 Issue 3, p689
Academic Journal
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.


Related Articles

  • Measurements of electron-impact-dissociation cross section for neutral products. Sugai, Hideo; Toyoda, Hirotaka // AIP Conference Proceedings;2000, Vol. 500 Issue 1, p349 

    Neutral radicals play key roles in plasma CVD and etching, and a comprehensive data base is needed on electron-impact-dissociation cross section for neutral radical production. This paper reviews the direct measurement of the neutral dissociation cross section accomplished by using appearance...

  • Dissociative electron attachment to S2F10, S2OF10, and S2O2F10. Olthoff, J. K.; Stricklett, K. L.; Van Brunt, R. J.; Moore, J. H.; Tossell, J. A.; Sauers, I. // Journal of Chemical Physics;6/15/1993, Vol. 98 Issue 12, p9466 

    The absolute cross sections for dissociative electron attachment to the molecules S2F10, S2OF10, and S2O2F10 were measured in an electron transmission experiment. The corresponding negative-ion fragments were identified in a separate mass spectrometric measurement. For S2F10, the attachment of...

  • Kinetics of glycine arenesulfonylation in the water-propan-2-ol system. Kustova, T. P.; Kochetova, L. B.; Kalinina, N. V. // Russian Journal of General Chemistry;Jun2007, Vol. 77 Issue 6, p1040 

    The kinetics of the glycine N-acylation with 3-nitrobenzenesulfonyl chloride in the water-propan-2-ol mixture containing 40–80 wt% water was studied at 298 K. In going from the mixture containing 40% water to that containing 80% water, the arenesulfonylation rate constant increases by a...

  • Mass spectrometric determination of the percent dissociation of a high-density chlorine plasma. Gaddy, G.A.; Webb, S.F. // Applied Physics Letters;12/1/1997, Vol. 71 Issue 22, p3206 

    Presents results of a computer modeling of high-density chlorine plasma indicating the dissociation of gas. Confirmation of the dissociation by optical measurements; Use of a mass spectrometric method; Percent of molecular dissociation measured in an electron cyclotron resonance plasma.

  • Comparison of Dissociation of Ions in an Electrospray Source, or a Collision Cell in Tandem Mass Spectrometry. Bure, Corinne; Lange, Catherine // Current Organic Chemistry;Oct2003, Vol. 7 Issue 15, p1613 

    Electrospray ionization (ESI) is a soft ionization technique well suited for producing gas phase ions of biological macromolecules. The use of ESI when combined with collision-induced dissociation and mass analysis can give structural information about biomolecules. Traditionally, collisional...

  • Those Ions Know What They Are Doing, Part II. Busch, Kenneth L. // Spectroscopy;Sep2012, Vol. 27 Issue 9, p20 

    The article discusses ion dissociation methods and the various levels of dissociation as an internal energy's function in the protonated molecule. It says that ultimate test of the models' validity is a comparison of observed and predicted mass spectra. It examines the spectra recorded under...

  • Effect of kinetic and electronic energy on the reactions of Cr+ with H2, HD, and D2. Elkind, J. L.; Armentrout, P. B. // Journal of Chemical Physics;2/15/1987, Vol. 86 Issue 4, p1868 

    The reactions of atomic chromium ions with H2, HD, and D2 are examined using guided ion beam tandem mass spectrometry. The ground electronic state (6S) is found to react inefficiently. The thresholds of these data are analyzed to yield a 0 K bond dissociation energy for CrH+ of 1.37±0.09 eV...

  • Thermochemistry of gaseous ZrF, ZrF[sub 2], and ZrF[sub 3]. Hildenbrand, D.L.; Lau, K.H. // Journal of Chemical Physics;10/22/1997, Vol. 107 Issue 16, p6349 

    Reports on gaseous reaction equilibria involving the lower-valent zirconium-fluorine species by mass spectrometry and the derived reaction thermodynamics to evaluate the bond dissociation energies and standard enthalpies of formation. Correlation of the second and third law as an aide in the...

  • Dissociation of singly and multiply charged fullerenes: Emission of C[sub 4], or sequential... Foltin, M.; Echt, O.; Scheier, P.; Dunser, B.; Worgotter, R.; Muigg, D.; Matt, S.; Mark, T.D. // Journal of Chemical Physics;10/22/1997, Vol. 107 Issue 16, p6246 

    Reports on the achievement of direct mass spectrometric evidence that fullerene ions undergo unimolecular dissociation by sequential emission of two C2 units. Comparison of experimental and theoretical breakdown graphs; Reaction channels accessible to excited fullerenes; Importance of thermal...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics