Occurrence of S-(1,2-dicarboxyethyl)-cysteine at position 77 in mutant human lysozyme secreted by Saccharomyces cerevisiae.
A mutant human lysozyme P110, in which Val110 was replaced with Pro, was secreted by Saccharomyces cerevisiae; modification of the cysteine residue at position 77 was found in a purified mutant protein (P110-B) upon primary structure analysis. A peptide fragment containing 15 amino acid residues from Thr70 to Leu84 was obtained by proteolytic digestion of the protein and subsequently isolated by reverse-phase HPLC. This fragment was analyzed by high-resolution fast-atom-bombardment (FAB) mass spectrometry, which showed that 1,2-dicarboxyethyl group was attached to the thiol group of Cys77. This modification was confirmed by comparing it with a sample of chemically synthesized S-(1,2-dicarboxyethyl)-L-cysteine. It was found that the modification caused a disruption of the disulfide bond Cys77-Cys95 in the mutant molecule. These observations, plus structural considerations, suggest that Cys77 and Cys95 either remain uncrosslinked or the disulfide bond Cys77-Cys95, once formed, is opened during the final step in the folding of human lysozyme in vivo.
Protein Engineering Research Institute, Osaka, Japan.
M
M
Kikuchi
Y
Y
Taniyama
S
S
Kanaya
T
T
Takao
Y
Y
Shimonishi
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives / Q000032:analysis*; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009113:Muramidase / Q000032:analysis / Q000096:biosynthesis*; D009154:Mutation; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D010957:Plasmids; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D000596:Amino Acids; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009113:Muramidase; D003545:Cysteine