Expression, purification, and characterization of the yeast KEX1 gene product, a polypeptide precursor processing carboxypeptidase.
The Saccharomyces cerevisiae KEX1 gene encodes a protease with carboxypeptidase B-like activity involved in K1 and K2 killer toxins and alpha-factor (mating pheromone) precursors processing. The gene has been expressed using the baculovirus/insect cell system, and the KEX1 encoded protein (Kex1p) was purified to apparent homogeneity from detergent-solubilized membrane preparations of insect cells infected with the recombinant virus. The specific activity of the enzyme was enriched 126-fold as compared with the cell lysate, with a recovery of 29%. The NH2-terminal sequence of the purified active enzyme was identical to the predicted sequence after the removal of the signal peptide. This provides evidence that Kex1p, at least in insect cells, is not made as a proenzyme. The optimum pH for activity was 6.0, and the apparent pI value of the protein was below pH 3.0. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides: benzoyl-Phe-Ala-Arg (Km = 284 microM), furylacryloyl (fa)-Ala-Arg (Km = 516 microM), and fa-Ala-Lys (Km = 962 microM). The kinetic data obtained reveals that Kex1p preferentially cleaves the COOH-terminal arginine of peptides over the COOH-terminal lysine. Insect-derived Kex1p processes alpha-factor-Lys-Arg, its known natural substrate, to mature active alpha-factor, and this maturation event takes place in a sequential manner. Furthermore, the enzyme expresses very high affinity for the 15-amino acid-long peptide, alpha-factor-Lys-Arg (Ki = 22 microM), and somewhat lower affinity for the heptapeptides [Leu]enkephalin-Arg-Arg,-Arg-Lys, and [Met]enkephalin-Lys-Lys (Ki = 45, 57, and 81 microM, respectively). The data demonstrate that processing at the COOH terminus of the peptides tested stops after the cleavage of the Arg and/or Lys residues. The specificity of the enzyme for COOH-terminal basic amino acid residues of the peptides used in this study and its high affinity for alpha-factor-Lys-Arg confirms the role that Kex1p plays in polypeptide precursor processing in yeast.
Latchinian-Sadek
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Biotechnology Research Institute, National Research Council, Montréal, Québec, Canada.
Thomas
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D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002268:Carboxypeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004745:Enkephalins / Q000378:metabolism; D005800:Genes, Fungal; D007313:Insecta; D000072235:Mating Factor; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000378:metabolism; D010452:Peptide Biosynthesis; D010455:Peptides; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D014162:Transfection
D002413:Cations, Divalent; D004745:Enkephalins; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D000072235:Mating Factor; D002268:Carboxypeptidases; C078874:carboxypeptidase D