Radioaffinity labelling of β-d-galactosidase from Escherichia coli with [14C]-glycerol, mediated through covalently bound 2,6-anhydro-1-deoxy-d-galacto-hept-1-enitol
✍ Scribed by Jochen Lehmann; Berit Schwesinger
- Publisher
- Elsevier Science
- Year
- 1982
- Tongue
- English
- Weight
- 281 KB
- Volume
- 110
- Category
- Article
- ISSN
- 0008-6215
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✦ Synopsis
The preceding paper' describes the application of 2,6 : 3,4-dianhydro-ldeoxy-D-&lo-hept-1-enitol (1) as a potential affinity label for the /?-D-galactosidase from E. co&. Compound 1, although having a reasonable Ki value of 2lmar, does not link exclusively to the substrate binding site of the enzyme, but links also to other groups of the protein molecule that have no influence on the catalytic activity. Thus 3H-labelled 1 attaches itself to the tetrameric enzyme in the molar ratio of N 48 : 1. This chemically modified enzyme, in comparison with an untreated blank, had lost less than 7 % of its activity in a calorimetric assay with an o-nitrophenyl j?-u-galactoside* (niphegal).
Discrimination between true affinity labelling and random labelling may be possible if the covalently attached label at the substrate-binding site would, because of its very presence there, undergo exclusively a secondary reaction catalyzed by the enzyme itself.
RESULTS AND DISCUSSION
2,6-Anhydro-1-deoxy-Dgalacto_hept-l-enitol ( 2) is converted into l-deoxy-Dgalacto-heptulose (3), or, in the presence of the acceptor, glycerol', into glycerol-l-y1 I-deoxy$-D-g&cto-heptulopyranoside (4) by j?-D-galactosidase in phosphate buffer.
As it could be demonstrated' that 2,6 : 3,4-dianhydro-l-deoxy-u-taio-hept-lenitol(1) becomes covalently attached to the substrate-binding site of j3-D-galactosidase, probably through its strongly electrophilic C-3 to form 2-E, there should be no *Complete loss of activity in tbe nipbegal assay can only be reached when the enzyme has been treated with extremely high concentrations of 1 (~-0.5~). These concentrations also cause Edhaustive, random labelling of the protein'.