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Pro Region C-Terminus:Protease Active Site Interactions Are Critical in Catalyzing the Folding of R-Lytic Protease
 

Summary: Pro Region C-Terminus:Protease Active Site Interactions Are Critical in Catalyzing
the Folding of R-Lytic Protease
Reuben J. Peters, Andrew K. Shiau, Julie L. Sohl, D. Eric Anderson, Gale Tang,| Joy L. Silen, and
David A. Agard*,
The Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, Graduate Group in Biophysics, and
Department of Physiology and Biochemistry, UniVersity of California, San Francisco, California 94143, and
Affymax Research Institute, 4001 Miranda AVenue, Palo Alto, California 94304
ReceiVed April 20, 1998; ReVised Manuscript ReceiVed June 26, 1998
ABSTRACT: R-Lytic protease is encoded with a large (166 amino acid) N-terminal pro region that is required
transiently both in vivo and in vitro for the correct folding of the protease domain [Silen, J. L., and
Agard, D. A. (1989) Nature 341, 462-464; Baker, D., et al. (1992) Nature 356, 263-265]. The pro
region also acts as a potent inhibitor of the mature enzyme [Baker, D., et al. (1992) Proteins: Struct.,
Funct., Genet. 12, 339-344]. This inhibition is mediated through direct steric occlusion of the active
site by the C-terminal residues of the pro region [Sohl, J. L., et al. (1997) Biochemistry 36, 3894-3904].
Through mutagenesis and structure-function analyses we have begun to investigate the mechanism by
which the pro region acts as a single turnover catalyst to facilitate folding of the mature protease. Of
central interest has been mapping the interface between the pro region and the protease and identifying
interactions critical for stabilizing the rate-limiting folding transition state. Progressive C-terminal deletions
of the pro region were found to have drastic effects on the rate at which the pro region folds the protease
but surprisingly little effect on inhibition of protease activity. The observed kinetic data strongly support

  

Source: Agard, David - Department of Biochemistry and Biophysics, University of California at San Francisco

 

Collections: Biotechnology; Biology and Medicine