title: Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme.
abstract: Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95. Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast. A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme. On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly. These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable. The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77. The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.
authors.affiliation: Third Research Department, Protein Engineering Research Institute, Osaka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Taniyama
authors.forename: Y
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: R
authors.initials: R
authors.lastname: Kuroki
authors.forename: M
authors.initials: M
authors.lastname: Kikuchi
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010434:Pepsin A; D010449:Peptide Mapping; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D004220:Disulfides; D009113:Muramidase; D010434:Pepsin A; D003545:Cysteine