Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli.
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Herrchen M, Legler G
Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli.
Eur J Biochem. 1984 Feb 1;138(3):527-31.
- PubMed ID
- 6420154 [ View in PubMed]
- Abstract
[3H] Conduritol C cis-epoxide (1,2-anhydro-epi-inositol, I) was synthesized as an active-site-directed inhibitor for lacZ beta-galactosidase from Escherichia coli. A considerable kinetic isotope effect was noted in the reduction by [3H]NaBH4 of the p-benzoquinone-derived precursor for I. Complete loss of beta-galactosidase activity occurred on incorporation of 4 mol I/mol beta-galactosidase tetramer. The inhibitor was very labile in the denatured enzyme at pH greater than 8, implying the formation of an ester bond between I and a carboxylate at the active site. The radioactive material released from the labeled enzyme was identified as allo-inositol. The stereochemistry of the expoxide reaction (trans-diaxial ring opening) is thus the same as for beta-glucosidases with the corresponding epoxides. The binding site for I was identified as Glu-461 by the isolation and partial sequence analysis of a radioactive octapeptide from the cyanogen bromide and pepsin fragments of the labeled enzyme. A failure to determine the N-terminal amino acid of the labeled peptide is ascribed to the great reactivity of the esterified gamma-carboxyl group of its N-terminal Glu-461 which causes rapid cyclisation of this residue to pyroglutamate, even under weakly basic conditions. The participation of the carboxylate of Glu-461 in catalysis is discussed.