AUTOCATALYTIC PROCESSING OF PRO-PAPAYA PROTEINASE-IV IS PREVENTED BY CROWDING OF THE ACTIVE-SITE CLEFT

The DNA coding for pro-papaya proteinase IV (PPIV) has been cloned and expressed in Escherichia coil. Heterologous expression of the protein , followed by refolding in vitro, yields an enzymatically active pro-e nzyme which fails to autodigest to form the mature protein, Mutagenesi s of the active site of papain to simulate that of PPIV yields a pro-e nzyme which also fails to autoactivate. Complementary mutagenesis of t he pro-region/mature boundary of PPIV, to introduce its own substrate recognition sequence, has, however, produced a pro-enzyme that will au tocatalytically cleave. This is the first report of enzymatic activity in a recombinant pro-cysteine proteinase, and the first time that suc h a protein has been shown to fail to autocatalytically cleave because of its stringent substrate specificity.