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. Copyright © 1990, Wiley Blackwell. All rights reserved