Article

Role of sulfhydryl groups in the function of glucosidase I from mammary gland

Department of Animal Sciences, University of Maryland, College Park 20742.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/1993; 268(9):6445-52.
Source: PubMed

ABSTRACT

Glucosidase I initiates the processing of asparagine-linked glycoproteins by excising the distal alpha 1,2-linked glucosyl residue from the Glc3Man9GlcNAc2 oligosaccharide, soon after its en bloc transfer from the lipid-linked donor to the nascent polypeptide. 1-Deoxynojirimycin, an analog of D-glucose, is a potent competitive inhibitor of the enzyme. Sulfhydryl-seeking reagents also strongly inhibit the enzyme, implying the involvement of an -SH group in its activity. To test this hypothesis, glucosidase I was purified from the rat mammary gland and its active site was loaded with 1-deoxynojirimycin, to protect such a group(s), while -SH groups on the remaining surface of the enzyme were blocked with N-ethylmaleimide or para-chloromercuriphenylsulfonic acid. Deoxynojirimycin was removed by dialysis to expose the active site -SH group(s). This group(s) was then tagged with 3-(N-maleimidopropionyl)biocytin (MPB) and detected with 125I-streptavidin on Western blots. A series of experiments is presented to show that indeed a critical -SH group(s) is located within the catalytic site of the enzyme. Additionally, the enzyme also possesses one or more sulfhydryls and disulfide bonds in its primary structure. The experimental approach outlined here should apply to identify reactive sulfhydryl groups in other catalytically active proteins.

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    • "α-Glucosidase I catalysis, like other glycosidases, is controlled by carboxylic acid residues (Koshland, 1953), as previously demonstrated by selective chemical modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Dhanawansa et al., 2002). Other residues, including Arg, Cys, and Trp, were reported to be likely participants in the binding site of mammalian α-glucosidase I based on chemical modification (Pukazhenthi et al., 1993; Romaniouk and Vijay, 1997). Also, mutated α-glucosidase I isolated from a patient with congenital disorder of glycosylation type IIb showed that together, Arg 486 Thr and Phe 652 Leu substitutions largely inactivated the enzyme (De Praeter et al., 2000; Volker et al., 2002). "
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    • "The structural organization of glucosidase I gene shows a relationship with the functional domains of the enzyme. The first exon codes for the cytoplasmic tail and transmembrane domain of the enzyme, the second and third exons encode the amino acid residues 116 through 192 and 193 through 257, respectively, whereas the fourth exon encodes the putative catalytic domain containing both the active and the glycosylation sites (Shailubhai et al., 1991; Pukazhenthi et al., 1993; Romaniouk and Vijay, 1997). At present, no clear function can be assigned to second and third exons. "
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