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Protein Engineering vol. 9 no. 6 pp. 525-529, 1996
© 1996 Oxford University Press

RESEARCH-ARTICLE

Autocatalytic processing of pro-papaya proteinase IV is prevented by crowding of the active-site cleft

Kenneth C. Baker1, Mark A J. Taylor2, Nicola J. ummings, Maria-Antonieta Tu ón3, Kathryn A. Worboys and Ian F. Connerton

Department of Food Macromolecular Science, Institute of Food Research, Reading Laboratory Earley Gate, Whiteknights Road, Reading RG6 2EF, UK

2To whom correspondence should be addressed

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. Complementarymutagenesis 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.

Keywords: cysteine proteinase//enzyme activation//papaya proteinase IV//pro-enzyme activity

Received December 8, 1995; revised January 31, 1996; accepted February 16, 1996.


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