title: Autocatalytic processing of pro-papaya proteinase IV is prevented by crowding of the active-site cleft.
abstract: The DNA coding for pro-papaya proteinase IV (PPIV) has been cloned and expressed in Escherichia coli. Heterologous expression of the protein, followed by refolding in vitro, yields an enzymatically active pro-enzyme which fails to autodigest to form the mature protein. Mutagenesis of the active site of papain to simulate that of PPIV yields a proenzyme which also fails to autoactivate. Complementary mutagenesis of the pro-region/mature boundary of PPIV, to introduce its own substrate recognition sequence, has, however, produced a pro-enzyme that will autocatalytically cleave. This is the first report of enzymatic activity in a recombinant pro-cysteine proteinase, and the first time that such a protein has been shown to fail to autocatalytically cleave because of its stringent substrate specificity.
authors.affiliation: Department of Food Macromolecular Science, Institute of Food Research, Reading Laboratory, UK.
authors.forename: K C
authors.initials: KC
authors.lastname: Baker
authors.forename: M A
authors.initials: MA
authors.lastname: Taylor
authors.forename: N J
authors.initials: NJ
authors.lastname: Cummings
authors.forename: M A
authors.initials: MA
authors.lastname: Tuñón
authors.forename: K A
authors.initials: KA
authors.lastname: Worboys
authors.forename: I F
authors.initials: IF
authors.lastname: Connerton
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005638:Fruit / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010206:Papain / Q000737:chemistry / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D004792:Enzyme Precursors; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases; D010206:Papain; C050874:glycyl endopeptidase