title: Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli.
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.
mesh_terms: D001665:Binding Sites; D001894:Borohydrides; D002264:Carboxylic Acids / Q000032:analysis*; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005696:Galactosidases / Q000302:isolation & purification*; D007294:Inositol / Q000031:analogs & derivatives / Q000378:metabolism; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
chemical_list: D001894:Borohydrides; D002264:Carboxylic Acids; D007294:Inositol; C025364:sodium borohydride; D005696:Galactosidases; D001616:beta-Galactosidase; C011423:conduritol epoxide