SELECTIVE ELONGATION OF THE OLIGOSACCHARIDE ATTACHED TO THE 2ND POTENTIAL GLYCOSYLATION SITE OF YEAST EXOGLUCANASE - EFFECTS ON THE ACTIVITY AND PROPERTIES OF THE ENZYME

Three exoglucanases (Exgs), ExgIa, ExgIb and Exg(325), are secreted by Saccharomyces cerevisiae cells. They share a common protein portion w ith two potential glycosylation sites (sequons) but differ in the amou nt of N-linked carbohydrate [Basco, R. D., Munoz, M. D., Hernandez, L. M., Vaquez de Aldana, C. and Larriba, G. (1993) Yeast 9, 221-234]. Ex gIb contains two short oligosaccharides attached to asparagines (Asn) 165 and 325 of the primary translation product [Hernandez, L. M., Oliv ero, I., Alvarado, E. and Larriba, G. (1992) Biochemistry 31, 9823-983 1]. Exg(325) carries a single, short oligosaccharide bound to Asn(325) whereas ExgIa has at least one large oligosaccharide, since it has no t been produced by mutant mnn9. To address the question of the origin of ExgIa, both sequons were individually mutated by substituting Gin f or Asn. An ExgIa-like isoenzyme was still secreted by mutant Exg,,, bu t not by mutant Exg(325). Additional studies on sequential deglycosyla tion of ExgIa with endo-beta-N-acetylglucosaminidase H (endo H), the s usceptibility of both oligosaccharides to the endoglycosidase, and ana lysis of the presence of GlcNAc at both asparagine residues after tota l deglycosylation with endo H, indicated that ExgIa contained two olig osaccharides, a short one bound to Asn(165) and a large one bound to A sn(325), and, accordingly, originated from ExgIb. The elongation of th e second oligosaccharide did not result in a higher stability towards thermal inactivation or unfolding, or in an increased resistance to pr oteases as compared with ExgIb; however, the affinity of the enzyme to wards laminarin decreased by 50%. This site-specific elongation occurr ed in the oligosaccharide that was less susceptible to endo H, indicat ing that these properties are determined by different conformational c onstraints.