PMID:1350782
| Citation |
Gebler, JC, Aebersold, R and Withers, SG (1992) Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli. J. Biol. Chem. 267:11126-30 |
|---|---|
| Abstract |
The covalent intermediate formed during catalysis by the lac Z beta-galactosidase from Escherichia coli can be trapped by reaction of the enzyme with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside, thereby inactivating the enzyme. Kinetic parameters for this inactivation process with the holo- and apo-enzymes have been determined. The intermediate so formed turns over only very slowly (t1/2 = 11.5 h) resulting in reactivation of the enzyme. The nucleophilic amino acid involved has been identified as Glu-537 by using a tritium-labeled inactivator to label the enzyme, then cleaving the labeled protein into peptides and purifying and sequencing the labeled peptide. This residue is conserved in five homologous beta-galactosidases and is different from that (Glu-461) proposed to be the nucleophile (Herrchen, M., and Legler, G. (1984) Eur. J. Biochem. 138, 527-531) on the basis of affinity labeling studies with conduritol C cis-epoxide. A role for glutamic acid residue 461 as the acid/base catalyst is proposed and justified. |
| Links | |
| Keywords |
Amino Acid Sequence; Binding Sites; Catalysis; Enzyme Activation; Escherichia coli/enzymology; Fucose/metabolism; Glutamates/genetics; Glutamates/metabolism; Glutamic Acid; Glycosides/metabolism; Kinetics; Molecular Sequence Data; Nitrophenylgalactosides/metabolism; beta-Galactosidase/antagonists & inhibitors; beta-Galactosidase/genetics; beta-Galactosidase/metabolism |
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