title: Affinity labelling with a deaminatively generated carbonium ion. Kinetics and stoicheiometry of the alkylation of methionine-500 of the lacZ beta-galactosidase of Escherichia coli by beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.
abstract: 1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene is an active-site-directed irreversible inhibitor of Mg2+-bound and Mg2+-free lacZ beta-galactosidase from Escherichia coli. 2. The Mg2+-enzyme binds the inhibitor more tightly but the complex then decomposes less rapidly than is the case with Mg2+-free enzyme. 3. Loss of enzyme activity is a linear function of the fraction of enzyme protomers to which are attached beta-D-galactopranosyl[14C]methyl residues: complete inactivation of fully active enzyme results in incorporation of 0.91 equivalent of carbohydrate label per enzyme protomer. 4. When the beta-galactopyranosylmethyl cation is generated in the active site of Mg2+-enzyme, it is captured essentially completely by the protein, but in the active site of Mg2+-free enzyme it is only captured with an efficiency of 25%. 5. Labelled enzyme was carboxymethylated and digested with trypsin; acidic hydrolysis of the isolated tryptic peptide, and field-desorption mass spectrometry of the isolated radioactive derivative, showed it to be 2,5-dioxo-3[2-(beta-D-galactopyranosylmethylthio)ethyl]-1,6-trimethylenepiperazine. 6. This is considered to have arisen from labelling of the sulphur atom of a methionine residue adjacent to a proline residue. 7. The complete amino acid sequence of the molecule [Fowler & Zabin (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1507-1510] enables the labelled methionine residue to be identified as either Met-421 or Met-500. 8. Sequence data [Fowler, Zabin, Sinnott & Smith (1978) J. Biol. Chem. in the press] show the site of attack to be Met-500.
mesh_terms: D000345:Affinity Labels; D000478:Alkylation; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005696:Galactosidases / Q000037:antagonists & inhibitors*; D005697:Galactosides / Q000494:pharmacology*; D006027:Glycosides / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium; D008715:Methionine; D014226:Triazenes / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors*
chemical_list: D000345:Affinity Labels; D005697:Galactosides; D006027:Glycosides; D014226:Triazenes; C018547:beta-D-galactopyranosylmethyl-4-nitrophenyltriazene; D008715:Methionine; D005696:Galactosidases; D001616:beta-Galactosidase; D008274:Magnesium