Recombinant pro-regions from papain and papaya proteinase IV are selective high affinity inhibitors of the mature papaya enzymes

Proteolytic enzymes require the presence of their proregionsfor correct folding. Of the four proteolytic enzymes from Caricapapaya, papain and papaya proteinase IV (PPIV) have 68% sequenceidentity. We find that their proregions are even more similar,exhibiting 73.6% identity. cDNAs encoding the pro-regions ofthese two proteinases have been expressed in Escherichia coliindependently from their mature enzymes. The recombinant pro-regionsof papain and PPIV have been shown to be high affinity inhibitorsof all four of the mature native papaya cysteine proteinases.Their inhibition constants are in the range 10^–6–;10^–;9M. PPIV was inhibited two to three orders of magnitude lesseffectively than papain, chymopapain and caricain. The pro-regionof PPIV, however, inhibited its own mature enzyme more effectivelythan did the proregion of papain. Alignment of the sequencesof the four papaya enzymes shows that there is a highly variablesection towards the C-terminal of the pro-region. This regionmay therefore confer selectivity to the pro-regions for theindividual proteolytic enzymes.