Abstract
(4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-beta-d-galactopyranoside was synthesized and successfully tested on beta-galactosidases from Xanthomonas manihotis (Wong-Madden, S. T., and Landry, D. Glycobiology (1995) 5, 19-28 and Taron, C. H., Benner, J. S., Hornstra, L. J., and Guthrie, E. P. (1995) Glycobiology 5, 603-610), Escherichia coli (Jacobson, R. H., Zhang, X. J., DuBose, R. F., and Matthews, B. W. (1994) Nature 369, 761-766), and Bacillus circulans (Fujimoto, H., Miyasato, M., Ito, Y., Sasaki, T., and Ajisaka, K. (1988) Glycoconj. J. 15, 155-160) for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from beta-galactosidases. It was demonstrated by using X. manihotis beta-galactosidase that the Arg-58 residue, which is located within a sequence of 56IPRAYWKD63, was labeled by nucleophilic attack of the guanidinyl group. This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42 (Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., and Wakagi, T. (2002) J. Mol. Biol. 322, 79-91). A catalytic glutamic acid (Glu-537) of E. coli beta-galactosidase was proved to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity. Similarly, a Glu-259 in 257TLEE260 was selectively labeled using B. circulans beta-galactosidase, indicating that Glu-259 is one of the nucleophiles in the active site. The present method can be readily extended to other glycosidases and should greatly aid the high throughput proteomics of many glycoside hydrolases showing both retaining- and inverting-type mechanisms.
Highlights
(4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)--D-galactopyranoside was synthesized and successfully tested on -galactosidases from Xanthomonas manihotis
Synthesis—Fluorescence-labeled suicide substrate 4 for high throughput proteomics of -galactosidases was prepared from a known 2-difluoromethyl-4-nitro-phenyl 2,3,4,6-tetra-O-acetyl-D-galactopyranoside by de-O-acetylation, reduction of the 4-nitro group, and a subsequent N-dansylation reaction as indicated in Scheme 1
The matrix-assisted laser desorption ionization (MALDI)-TOF mass spectra of the peptides readily purified by the antidansyl antibody column showed clear and simple signals corresponding to the labeled peptide fragments
Summary
Pluckthun and co-workers [33] have succeeded in trapping a catalyst with alkaline phosphatase activity by using a similar mechanism-based chemical selection strategy In adopting this principle, we chose fluorescent labeling of GH followed by peptide fingerprinting by mass spectroscopy in the high throughput proteomics of GHs. When a glycosidase is expressed and produced using common bacterial systems, identification of the target protein in a cell lysate, a complex mixture of proteins and other biomolecules, requires a rather tedious sequence of blotting, digestion, and staining procedures prior to mass spectroscopic analyses [17]. We thought that irreversible fluorescence labeling of GHs would facilitate isolation and identification of labeled active site peptides as well as labeled GHs. We demonstrate that a mechanism-based tagging of -galactosidases with a fluorescence-labeled suicide substrate (Fig. 1) greatly expedited both an efficient chromatographic separation and an accurate sequencing of the labeled peptides by means of a tandem mass spectroscopy (MALDI-TOF/TOF)
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