Identification of an essential carboxylate group at the active site of lacZβ‐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 P-galactosidase from Escherichia coli. A considerable kinetic isotope effect was noted in the reduction by [3H]NaBH, of the p-benzoquinone-derived precursor for I.Complete loss of fl-galactosidase activity occurred on incorporation of 4 mol I/mol fl-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 P-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 Nterminal amino acid of the labeled peptide is ascribed to the great reactivity of the esterified 11-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.At least two functional groups are thought to be essential for catalysis by P-galactosidase from Escherichia coli: an acidic group donating a proton to the galactosyl oxygen and a carboxylate that aids bond breaking by partially neutralizing a positive charge on C-1 in the transition state and formation of an a-galactosyl ester intermediate [I]. Evidence for these groups is mainly indirect. The acid is inferred from pHdependence of k,,, [2] and from the influence of Mg2+ on the hydrolysis of 0-and N-galactosides [3] and on the tight binding of basic N-galactosyl derivatives [4]. A possible candidate for this acid is T~r -5 0 3~ which is adjacent to a Met residue that can be labeled by the active-site-directed inhibitors N-bromoacetyl-P-galactosylamine [7] and P-galactosylmethyl-p-nitrophenyltriazene [8]. The participation of a carboxylate is founded on kinetic considerations, on the ability of conduritol C cis-exposide (I) to act as an active-site-directed inhibitor [9] and on the formation of an alkali-labile 2-deoxygalactosyl-enzyme intermediate on reaction with D-We now report the identification of the binding site for I as Glu-461 and give evidence that this site represents the carboxylate group taking part in catalyis.The numbering of the amino acid residues has been changed from that given by Fowler and Zabm [5] to accomodate two additional amino acids as required by the base sequence of the lacZ gene [6].Part of this paper is taken from the doctoral thesis of M. Herrchen (University of Cologne, 1983).Ahhreaiations. TLC, thin-layer chromatography. Enzyme. P-Galactosidase, P-D-galactoside galactohydrolase (EC 3.2.1.23) MATERIALS A N D METHODS Specific radioactivity o j f H] NaBH4[3H]NaBH,, 0.5 -2 pmol in 0.1 ...

Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco.

“…The authors concluded that the fusions were being degraded by translocation to the vacuole, but our observations indicate that another explanation for the loss of enzymatic activity may have been Nglycosylation of GUS. It is noteworthy that the putative active site of GUS, which can be identified by its close similarity to the active site of /3-galactosidase (Herrchen Legler, 1984), is located between the two putative Nglycosylation sites, at amino acid residues 401 to 413.…”

“…The authors concluded that the fusions were being degraded by translocation to the vacuole, but our observations indicate that another explanation for the loss of enzymatic activity may have been Nglycosylation of GUS. It is noteworthy that the putative active site of GUS, which can be identified by its close similarity to the active site of /3-galactosidase (Herrchen Legler, 1984), is located between the two putative Nglycosylation sites, at amino acid residues 401 to 413.The observation that active GUS is formed after tunicamycin treatment means that GUS has assembled into an active tetramer in the lumen of the ER. This is significant because severa1 important foreign proteins that are currently being expressed in plants need to be modified and assembled into active molecules in the ER.…”

Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine‐linked oligosaccharides

“…Glucosidase I resembles fl-glucosidase A, from Aspergillus wvntii in being strongly inhibited by substrate-related cations [19]. Glucosidase IT, on the other hand, is similar to b-glucosidase from almonds [20] and [j-galactosidase from Escherichia coli [21], which show strong inhibition with basic glycosyl derivatives, but not with closely related cationic ones.…”

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

“…The substrate has to move about 3 Å deeper into the active site (E d Gal-OR) for catalysis to begin. The roles of Glu461 and Glu537 as the acid catalyst and the nucleophile, respectively, have been well established by chemical modification, 70,71 site-directed mutagenesis, [72][73][74] and structural studies. 29 Deep-site binding is visualized by the binding of galactose to the free enzyme, 29 in which the galactose stacks with Trp568 [ Fig.…”