In vitro, procathepsin D is activated to pseudocathepsin D by incubation at
low pH. To investigate the mechanism of this activation, recombinant human
procathepsin D and two mutants were generated in a baculovirus expression
system. One mutant carried a point mutation within the catalytic domain, wh
ich resulted in a catalytically inactive enzyme form (D77A). The other carr
ied a point mutation within the propeptide, which prevented activation by p
rocessing at the 'autoproteolysis-site' (L26P). Neither mutant is capable o
f processing itself to form pseudocathepsin D, and L26P is not able to proc
ess D77A. Despite the inability of L26P to cleave either its own or a wild-
type prosequence, it did exhibit activity against a synthetic peptide subst
rate. The ability of intact precursor (zymogen) to cleave a peptide, but no
t a protein substrate, offers new insights into the mechanism of inhibition
by the propeptide. Mature cathepsin D can process the inactive D77A mutant
to the pseudoform, demonstrating that processed species are capable of cle
aving zymogen molecules in an intermolecular interaction. In addition, kine
tic studies provide evidence for a two-phase mechanism for the conversion o
f procathepsin D to pseudocathepsin D, one phase where the first molecules
of pseudocathepsin D are formed at a low rate and a second phase where the
process is autocatalytically accelerated by newly formed pseudocathepsin D
molecules. Finally, with the help of the mutants L26P and D77A it was obser
ved that at least two additional proteinase activities, found in conditione
d media from insect cell culture, are capable of activating procathepsin D
by cleaving it within the proregion. This observation suggests that there a
re likely to be multiple proteinases in the extracellular matrix that are c
apable of activating procathepsin D, thereby triggering the second autocata
lytic phase. This may also be important for solid tumors, where the presenc
e of cathepsin D has been correlated with tumor growth and invasion.