title: Role of carbohydrate moiety in carboxypeptidase Y: structural study of mutant enzyme lacking carbohydrate moiety.
abstract: To study the roles of the carbohydrate moiety in the function of carboxypeptidase Y, asparagine residues at 13, 87, 168, and 368, the four-consensus N-linked glycosylation sites, were altered to alanine with site-directed mutagenesis. The mutant enzyme of 51 kDa completely lost the carbohydrate moiety which was present in the 61-kDa wild-type enzyme. Structural studies of the mutant enzyme showed that it maintained the native-like structure; hydrolytic activity, and substrate specificity of the mutant enzyme analogous to those of the wild-type enzyme. Susceptibility of the mutant enzyme toward proteolysis and pressure denaturation was reduced by 10-20%. It is concluded that the carbohydrate moiety functions to maintain the structural integrity of the enzyme under stressed.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Shimizu
authors.forename: H
authors.initials: H
authors.lastname: Ueno
authors.forename: R
authors.initials: R
authors.lastname: Hayashi
mesh_terms: D002241:Carbohydrates / Q000737:chemistry*; D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D043402:Cathepsin A; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
chemical_list: D002241:Carbohydrates; D002268:Carboxypeptidases; D043402:Cathepsin A