Overexpression and Functional Characterization of a Serine Carboxypeptidase Inhibitor (Ic) from Saccharomyces cerevisiae

• Published in: Journal of biochemistry (Tokyo) Vol. 132; no. 6; pp. 967 - 973
• Main Authors: Mima, Joji, Suzuki, Hidekazu, Takahashi, Mieko, Hayashi, Rikimaru
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.12.2002
• Subjects: carboxypeptidase inhibitor; carboxypeptidase Y; Cathepsin A - antagonists & inhibitors; Cathepsin A - genetics; Cathepsin A - metabolism; Circular Dichroism; Dithionitrobenzoic Acid - metabolism; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; Macromolecular Substances; Molecular Weight; overexpression; p-Chloromercuribenzoic Acid - metabolism; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; proteinase-inhibitor complex; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfhydryl Reagents - metabolism
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a003311

Carboxypeptidase Y (CPY) inhibitor, Ic, a cytoplasmic inhibitor of vacuolar proteinases in yeast, Saccharomyces cerevisiae, was purified by means of a high-level expression system using a proteinase-deficient strain, BJ2168, and an expression vector with the promoter GALI. The purified Ic exists as a monomeric βprotein in solution with a molecular weight of 24, 398.4 as determined by gel filtration chromatography, MALDI-TOF mass spectrometry, and far-UV CD spectroscopy. The acetylated N-terminal methionine residue is the sole posttranslational modification. Ic specifically inhibits both the pepti-dase and anilidase activities of CPY with inhibitor constants (K1) of approximately 1.0 × 10−8 M. The chemical modification of Ic with sulfhydryl reagents indicated that it lacks disulfide bonds and has two free SH groups, which are responsible, not for the inhibitory function, but, apparently, for the folding of the overall structure. The formation of a complex of Ic with CPY was highly specific, as evidenced by no detectable interaction with pro-CPY. Chemical modification studies of the CPY-Ic complex with specific reagents demonstrated that the catalytic Ser146 and SI substrate-binding site of CPY are covered in the complex.