Identification of an essential carboxylate group at the active site of lacZ β‐galactosidase from Escherichia coli

[ 3 H] Conduritol C cis ‐epoxide (1,2‐anhydro‐ epi ‐inositol, I) was synthesized as an active‐site‐directed inhibitor for lacZ β‐galactosidase from Escherichia coli. A considerable kinetic isotope effect was noted in the reduction by [ 3 H]NaBH 4 of the p ‐benzoquinone‐derived precursor for I. Complete loss of β‐galactosidase activity occurred on incorporation of 4 mol I/mol β‐galactosidase tetramer. The inhibitor was very labile in the denatured enzyme at pH < 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 β‐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 γ‐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.