Role of carbohydrate moiety in carboxypeptidase Y: structural study of mutant enzyme lacking carbohydrate moiety.
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.
Shimizu
H
H
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Ueno
H
H
Hayashi
R
R
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
D002241:Carbohydrates; D002268:Carboxypeptidases; D043402:Cathepsin A