Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli.
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.
Gebler
J C
JC
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Aebersold
R
R
Withers
S G
SG
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005643:Fucose / Q000378:metabolism; D005971:Glutamates / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid; D006027:Glycosides / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009598:Nitrophenylgalactosides / Q000378:metabolism; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D005971:Glutamates; D006027:Glycosides; C074857:2',4'-dinitrophenyl 2-deoxy-2-fluorogalactopyranoside; D009598:Nitrophenylgalactosides; D005643:Fucose; D018698:Glutamic Acid; D001616:beta-Galactosidase