Kc. Baker et al., AUTOCATALYTIC PROCESSING OF PRO-PAPAYA PROTEINASE-IV IS PREVENTED BY CROWDING OF THE ACTIVE-SITE CLEFT, Protein engineering, 9(6), 1996, pp. 525-529
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.