Folding pathway mediated by an intramolecular chaperone - Propeptide release modulates activation precision of pro-subtilisin

Propeptides of several proteases directly catalyze the protein folding reac tion. Uncatalyzed folding traps these proteases into inactive molten-globul e-like conformers that switch into active enzymes only when their cognate p ropeptides are added in trans. Although tight binding and proteolytic susce ptibility forces propeptides to function as single turnover catalysts, the significance of their inhibitory function and the mechanism of activation r emain unclear. Using pro-subtilisin as a model, we establish that precursor activation is a highly coordinated process that involves synchronized fold ing, autoprocessing, propeptide release, and protease activation. Our resul ts demonstrate that activation is controlled by release of the first free a ctive protease molecule. This triggers an exponential cascade that selectiv ely targets the inhibitory propeptide in the autoprocessed complex as its s ubstrate. However, a mutant precursor that enhances propeptide release can drastically reduce the folding efficiency by altering the synergy between i ndividual stages. Our results represent the first demonstration that propep tide release, not precursor folding, is the rate-determining step and provi des the basis for the proposed model for precise spatial and temporal activ ation that allows proteases to function as regulators of biological functio n.