Folding pathway mediated by an intramolecular chaperone: propeptide release modulates activation precision of pro-subtilisin.
Propeptides of several proteases directly catalyze the protein folding reaction. Uncatalyzed folding traps these proteases into inactive molten-globule-like conformers that switch into active enzymes only when their cognate propeptides are added in trans. Although tight binding and proteolytic susceptibility forces propeptides to function as single turnover catalysts, the significance of their inhibitory function and the mechanism of activation remain unclear. Using pro-subtilisin as a model, we establish that precursor activation is a highly coordinated process that involves synchronized folding, autoprocessing, propeptide release, and protease activation. Our results demonstrate that activation is controlled by release of the first free active protease molecule. This triggers an exponential cascade that selectively targets the inhibitory propeptide in the autoprocessed complex as its substrate. However, a mutant precursor that enhances propeptide release can drastically reduce the folding efficiency by altering the synergy between individual stages. Our results represent the first demonstration that propeptide release, not precursor folding, is the rate-determining step and provides the basis for the proposed model for precise spatial and temporal activation that allows proteases to function as regulators of biological function.
Yabuta
Y
Y
Department of Biochemistry and Molecular Biology, MRB631, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.
Takagi
H
H
Inouye
M
M
Shinde
U
U
D002384:Catalysis; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D013053:Spectrophotometry; D013381:Subtilisins / Q000737:chemistry; D013997:Time Factors
D004792:Enzyme Precursors; D018832:Molecular Chaperones; D010446:Peptide Fragments; D010455:Peptides; C057107:prosubtilisin; D010450:Endopeptidases; D013381:Subtilisins