AUTOCATALYTIC PROCESSING OR RECOMBINANT HUMAN PROCATHEPSIN-L - CONTRIBUTION OF BOTH INTERMOLECULAR AND UNIMOLECULAR EVENTS IN THE PROCESSING OF PROCATHEPSIN-L IN-VITRO

The autocatalytic processing of procathepsin L was investigated in vit ro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant pr ocathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrar y to recent reports, intermolecular processing of procathepsin L is po ssible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity o f cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic pr ocessing of procathepsin L can generate the natural mature form the en zyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by dec reasing the pH from 8.0 to 5.3. The influence of proenzyme concentrati on on the rate of processing indicates the existence of both unimolecu lar and biomolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Ci rcular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attribute d to intermolecular processing of the zymogen.