Processing of the papain precursor. The ionization state of a conserved amino acid motif within the Pro region participates in the regulation of intramolecular processing.
The cysteine protease papain is synthesized as a 40-kDa inactive precursor with a 107-amino-acid N-terminal pro region. Although sequence conservation in the pro region is lower than in the mature proteases, a conserved motif (Gly-Xaa-Asn-Xaa-Phe-Xaa-Asp-36, papain precursor numbering) was found within the pro region of cysteine proteases of the papain superfamily. To determinate the function to this conserved motif, we have mutagenized at random each of the 4 residues individually within the pro region of the papain precursor. Precursor mutants were expressed in yeast, screened according to their ability to be processed through either a cis or trans reaction, into mature active papain. Three classes of mutants were found. Non-functional propapain mutants of the first class are completely degraded by subtilisin indicating that they are not folded into a native state. Mutants of the second class were neutral with respect to cis and trans processing. The third class included mutants that mostly accumulated as mature papain in the yeast vacuole. They had mutations that had lost the negatively charged Asp-36 residues and a mutation that probably introduces a positive charge, Phe-38His. The precursor of the Phe-38His mutant could be recovered by expression in a vph1 mutant yeast strain which has a vacuolar pH of about 7. The Phe-38His propapain mutant has an optimum pH of autoactivation about one pH unit higher than the wild type molecule. These results indicate that the electrostatic status of the conserved motif participates in the control of intramolecular processing of the papain precursor.
Vernet
T
T
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec.
Berti
P J
PJ
de Montigny
C
C
Musil
R
R
Tessier
D C
DC
Ménard
R
R
Magny
M C
MC
Storer
A C
AC
Thomas
D Y
DY
D000595:Amino Acid Sequence; D016384:Consensus Sequence; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010206:Papain / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004792:Enzyme Precursors; D010455:Peptides; D010206:Papain