Processing of the papain precursor. Purification of the zymogen and characterization of its mechanism of processing.
The precursor of the cysteine protease papain has been expressed and secreted as propapain from insect cells infected with a recombinant baculovirus expressing a synthetic gene coding for prepropapain. This 39-kDa secreted propapain zymogen molecule is glycosylated and can be processed in vitro into an enzymatically active authentic papain molecule of 24.5 kDa (Vernet, T., Tessier, D.C., Richardson, C., Laliberté, F., Khouri, H. E., Bell, A. W., Storer, A. C., and Thomas, D. Y. (1990) J. Biol. Chem. 265, 16661-16666). Recombinant propapain was stabilized with Hg2+ and purified to homogeneity using affinity chromatography, gel filtration, and ion-exchange chromatographic procedures. The maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 65 degrees C and under reducing conditions. Precursor processing is inhibited by a variety of reversible and irreversible cysteine protease inhibitors but not by specific inhibitors of serine, metallo or acid proteases. Replacement by site-directed mutagenesis of the active site cysteine with a serine at position 25 also prevents processing. The inhibitor 125I-N-(2S,3S)-3-trans-hydroxycarbonyloxiran-2-carbonyl-L-tyrosine benzyl ester covalently labeled the wild type papain precursor, but not the C25S mutant, indicating that the active site is accessible to the inhibitor and is in a native conformation within the precursor. Based on biochemical and kinetic analyses of the activation and processing of propapain we have shown that the papain precursor is capable of autoproteolytic cleavage (intramolecular). Once free papain is released processing can then occur in trans (intermolecular).
Vernet
T
T
Genetic Engineering Section, National Research Council of Canada, Montréal, Québec.
Khouri
H E
HE
Laflamme
P
P
Tessier
D C
DC
Musil
R
R
Gour-Salin
B J
BJ
Storer
A C
AC
Thomas
D Y
DY
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005813:Genes, Synthetic; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010206:Papain / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014462:Ultrafiltration
D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C071653:propapain; D010206:Papain