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 precu rsor with a 107-amino-acid N-terminal pro region. Although sequence co nservation in the pro region is lower than in the mature proteases, a conserved motif (Gly-Xaa-Asn-Xaa-Phe-Xaa-Asp(-36), papain precursor nu mbering) was found within the pro region of cysteine proteases of the papain superfamily. To determinate the function to this conserved moti f, we have mutagenized at random each of the 4 residues individually w ithin the pro region of the papain precursor. Precursor mutants were e xpressed in yeast, screened according to their ability to be processed through either a cis or trans reaction, into mature active papain. Th ree 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 yeas t vacuole. They had mutations that had lost the negatively charged Asp (-36) residues and a mutation that probably introduces a positive char ge, Phe(-38)His. The precursor of the Phe(-38)His mutant could be reco vered by expression in a vph1 mutant yeast strain which has a vacuolar pH of about 7. The Phe(-38)His propapain mutant has an optimum pH of autoactivation about one pH unit higher than the wild type molecule. T hese results indicate that the electrostatic status of the conserved m otif participates in the control of intramolecular processing of the p apain precursor.