Affinity labelling with a deaminatively generated carbonium ion. Kinetics and stoicheiometry of the alkylation of methionine-500 of the lacZ β-galactosidase of Escherichia coli by β-d-galactopyranosylmethyl-p-nitrophenyltriazene

Abstract

.beta.-D-Galactopyranosylmethyl-p-nitrophenyltriazene is an active-site-directed irreversible inhibitor of Mg2+-bound and Mg2+-free lacZ .beta.-galactosidase from E. coli. The Mg2+-enzyme binds the inhibitor more tightly but the complex then decomposes less rapidly than is the case with Mg2+-free enzyme. Loss of enzyme activity is a linear function of the fraction of enzyme protomers to which are attached .beta.-D-galactopyranosyl[14C]methyl residues: complete inactivation of fully active enzyme results in incorporation of 0.91 equivalent of carbohydrate label/enzyme protomer. 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%. Labeled 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. This is considered to have arisen from labeling of the S atom of a methionine residue adjacent to a proline residue. The complete amino acid sequence of the molecule enables the labeled methionine residue to be identified as either Met-421 or Met-500. Sequence data show the site of attack to be Met-500.