Overexpression and functional characterization of a serine carboxypeptidase inhibitor (I(C)) from Saccharomyces cerevisiae.
Carboxypeptidase Y (CPY) inhibitor, I(C), 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 GAL1. The purified I(C) exists as a monomeric beta-protein in solution with a mole-cular 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. I(C) specifically inhibits both the peptidase and anilidase activities of CPY with inhibitor constants (K(i)) of approximately 1.0 x 10(-9) M. The chemical modification of I(C) 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 I(C) with CPY was highly specific, as evidenced by no detectable interaction with pro-CPY. Chemical modification studies of the CPY-I(C) complex with specific reagents demonstrated that the catalytic Ser146 and S1 substrate-binding site of CPY are covered in the complex.
Mima
Joji
J
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. mima@kais.kyoto-u.ac.jp
Suzuki
Hidekazu
H
Takahashi
Mieko
M
Hayashi
Rikimaru
R
D043402:Cathepsin A / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013439:Sulfhydryl Reagents / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid / Q000378:metabolism
D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D013439:Sulfhydryl Reagents; D020245:p-Chloromercuribenzoic Acid; D004228:Dithionitrobenzoic Acid; D043402:Cathepsin A