TITLE

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

AUTHOR(S)
Dennhart, Nicole; Letzel, Thomas
PUB. DATE
November 2006
SOURCE
Analytical & Bioanalytical Chemistry;Nov2006, Vol. 386 Issue 3, p689
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
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.
ACCESSION #
22458607

 

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