Proteolytic Performance Is Dependent on Binding Efficiency, Processivity, and Turnover: Single Protease Insights

Proteases are essential enzymes for a plethora of biological processes and biotechnological applications, e.g., within the dairy, pharmaceutical, and detergent industries. Decoding the molecular-level mechanisms that drive protease performance is the key to designing improved biosolutions. However, the direct dynamic assessment of the fundamental partial reactions of substrate binding and activity has proven to be a challenge with conventional ensemble approaches. We developed a single-molecule (SM) assay for the direct and parallel recording of the stochastic binding interaction of Savinase, a serine-type protease broadly employed in biotechnology, with casein, while synchronously monitoring proteolytic degradation of the substrate. This assay allowed us to elucidate how the overall activity of Savinase and its variants depends on binding efficiency, turnover, and activity per binding event. We identified three distinct binding states, with mutations primarily affecting the long-lived state, indicating that it contributes to the overall activity and suggesting a level of processivity in Savinase. These insights, inaccessible through conventional methods, provide valuable perspectives for engineering proteases with improved hydrolytic performance.