SPECIFIC-INHIBITION OF MATURE FUNGAL SERINE PROTEINASES AND METALLOPROTEINASES BY THEIR PROPEPTIDES

The function of the long propeptides of fungal proteinases is not know n. Aspergillus fumigatus produces a 33-kDa serine proteinase of the su btilisin family and a 42-kDa metalloproteinase of the thermolysin fami ly. These extracellular enzymes are synthesized as preproenzymes conta ining large amino-terminal propeptides. Recombinant propeptides were p roduced in Escherichia coli as soluble fusion proteins with glutathion e S-transferase or thioredoxin and purified by affinity chromatography . A. fumigatus serine proteinase propeptide competitively inhibited se rine proteinase, with a K-i of 5.3 x 10(-6) M, whereas a homologous se rine proteinase from A, flavus was less strongly inhibited and subtili sin was not inhibited. Binding of metalloproteinase propeptide from A, fumigatus to the mature metalloenzyme was demonstrated. This propepti de strongly inhibited its mature enzyme, with a K-i of 3 x 10(-9) M, w hereas thermolysin and a metalloproteinase from A. flavus were not inh ibited by this propeptide. Enzymatically inactive metalloproteinase pr opeptide complex could be completely activated by trypsin treatment. T hese results demonstrate that the propeptides of the fungal proteinase s bind specifically and inhibit the respective mature enzymes, probabl y reflecting a biological role of keeping these extracellular enzymes inactive until secretion.