Deep quantification of substrate turnover defines protease subsite cooperativity.

Substrate specificity determines protease functions in physiology and in clinical and biotechnological applications, yet quantitative cleavage information is often unavailable, biased, or limited to a small number of events. Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify >40,000 peptides from a complex, commercially available peptide mixture. By analyzing changes in substrate levels quantitatively instead of focusing on qualitative product identification through a binary classifier, we can reveal cooperative interactions within DPP4's active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related C. elegans DPF-3 (a DPP8/9-orthologue), and we relate the differences to the structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like the stabilization of GLP-1, a key DPP4 substrate used in the treatment of diabetes and obesity. Thus, qPISA offers a versatile approach for profiling protease and especially exopeptidase specificity, facilitating insight into enzyme mechanisms and biotechnological and clinical applications. Synopsis: To examine protease cleavage specificity quantitatively and understand the cooperativity among protease subsites, the authors develop a novel approach, qPISA, that models quantitative changes in input peptide levels upon digestion by a protease in vitro. Cooperative interactions within the active pockets of DPP IV family exopeptidases determine substrate specificity. Two closely related proteases exhibit overlapping yet distinguishable specificity profiles, explained by structural differences. The model reliably predicts experimentally determined peptide half-lives. The approach can instruct protein engineering approaches to modulate peptide half-lives. To examine protease cleavage specificity quantitatively and understand the cooperativity among protease subsites, the authors develop a novel approach, qPISA, that models quantitative changes in input peptide levels upon digestion by a protease in vitro. [ABSTRACT FROM AUTHOR]