PMID:6420154
| Citation |
Herrchen, M and Legler, G (1984) Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli. Eur. J. Biochem. 138:527-31 |
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| 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. |
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| Keywords |
Binding Sites; Borohydrides; Carboxylic Acids/analysis; Catalysis; Chemical Phenomena; Chemistry; Escherichia coli/enzymology; Galactosidases/isolation & purification; Inositol/analogs & derivatives; Inositol/metabolism; beta-Galactosidase/antagonists & inhibitors; beta-Galactosidase/isolation & purification |
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