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PMID:17485082
| Citation |
Shaikh, FA, Müllegger, J, He, S and Withers, SG (2007) Identification of the catalytic nucleophile in Family 42 beta-galactosidases by intermediate trapping and peptide mapping: YesZ from Bacillus subtilis. FEBS Lett. 581:2441-6 |
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| Abstract |
The mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-galactopyranoside (DNP2FGal) was used to inactivate the Family 42 beta-galactosidase (YesZ) from Bacillus subtilis via the trapping of a glycosyl-enzyme intermediate, thereby tagging the catalytic nucleophile in the active site. Proteolytic digestion of the inactivated enzyme and of a control sample of unlabeled enzyme, followed by comparative high-performance liquid chromatography and mass spectrometric analysis identified a labelled peptide of the sequence ETSPSYAASL. These data, combined with sequence alignments of this region with representative members of Family 42, unequivocally identify the catalytic nucleophile in this enzyme as Glu-295, thereby providing the first direct experimental proof of the identity of this residue within Family 42. |
| Links |
PubMed Online version:10.1016/j.febslet.2007.04.053 |
| Keywords |
Amino Acid Sequence; Bacillus subtilis/metabolism; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; Catalytic Domain; Cloning, Molecular; DNA Primers; Kinetics; Molecular Sequence Data; Peptide Mapping; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Spectrometry, Mass, Electrospray Ionization; Thermus/enzymology; beta-Galactosidase/chemistry; beta-Galactosidase/genetics; beta-Galactosidase/metabolism |
| edit table |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0004565: beta-galactosidase activity |
ECO:0000314: |
F |
Section 3.1. Enzyme kinetics (calculations not shown but described in methods): Kinetic parameters for the hydrolysis of pNPGal by wild type YesZ of kcat = 81 ± 4 s-1, Km = 3.0 ± 0.2 mM, kcat/Km = 27 ± 2 mM-1 s-1 were determined. |
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See also
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