Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 326, Issue 1, Pages 30-35Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2004.11.003
Keywords
quinone methide; hydrolases; protein tyrosine phosphatase; glucosidase; cysteine; labeling; proteome; activity probe; mechanism-based
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Use of activity probes has been demonstrated to be a powerful tool in modern chemical proteomic study. Previously we have designed and synthesized a series of mechanism-based activity probes that utilized quinone methide chemistry. Here, we characterized the trend of chemical reactivity for the reactive quinone methide intermediate 3 (QM-3) resulting from the latent trapping device. In a competition assay, the labeling of PTP1B by probe 1a was blocked by externally added cysteine without affecting the catalytic activity of the enzyme. Further sequencing analysis on the trypsin-digested peptides of probe la-labeled PTP1B using tandem mass spectrometry revealed that all six cysteine residues of PTP1B are capable of being modified by probe 1a. These results indicated that the sulfhydryl group of cysteine residue is the preferred nucleophile for the reactive QM-3. Our finding provides the first example in understanding the preferred amino acid residues modified by the reactive QM-3, which is also the key structural unit responsible for forming covalent bonds in many biochemical applications. (C) 2004 Elsevier Inc. All rights reserved.
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