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Glycobiology Advance Access published online on July 13, 2005
Glycobiology, doi:10.1093/glycob/cwj009
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org
Received May 18, 2005
Revised June 30, 2005
Accepted July 3, 2005

Article

Binding residues and catalytic domain of soluble Saccharomyces cerevisiae processing alpha-glucosidase I

Amirreza Faridmoayer 1 and Christine H. Scaman 1*

1 Food, Nutrition, and Health, University of British Columbia, Vancouver V6T 1Z4, Canada

* To whom correspondence should be addressed.
Christine H. Scaman, E-mail: cscaman{at}interchange.ubc.ca


  Abstract

{alpha}-Glucosidase I initiates the trimming of newly assembled N-linked glycoproteins in the lumen of the endoplasmic reticulum. Site-specific chemical modification of the soluble {alpha}- glucosidase I from yeast using diethylpyrocarbonate and tetranitromethane revealed that histidine and tyrosine are involved in the catalytic activity of the enzyme, as these residues could be protected from modification using the inhibitor deoxynojirimycin. Deoxynojirimycin could not prevent inactivation of enzyme treated with N-bromosuccinimide used to modify tryptophan residues. Therefore, the binding mechanism of yeast enzyme contains different amino acid residues compared to its mammalian counterpart.

Catalytically active polypeptides were isolated from endogenous proteolysis and controlled trypsin hydrolysis of the enzyme. A 37 kDa non-glycosylated polypeptide was isolated as the smallest active fragment from both digests, using affinity chromatography with inhibitor-based resins (N-methyl-N-5’-carboxypentyl- and N-5’-carboxypentyl- deoxynojirimycin). N-terminal sequencing confirmed that the catalytic domain of the enzyme is located at the C-terminus. The hydrolysis sites were between Arg521 and Thr522 for endogenous proteolysis and residues Lys524 and Phe525 for the trypsin generated peptide. This 37 kDa polypeptide is 1.9 times more active than the 98 kDa protein when assayed with the synthetic trisaccharide, {alpha}-D-Glc1,2{alpha}-D-Glc1,3{alpha}-D-Glc-O(CH2)8COOCH3, and is not glycosylated. Identification of this relatively small fragment with catalytic activity will allow mechanistic studies to focus on this critical region, and raises interesting questions about the relationship between the catalytic region and the remaining polypeptide.

Keywords: binding residues/catalytic domain/chemical modification/{alpha}-glucosidase I/Saccharomyces cerevisiae.
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