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 key to designing improved biosolutions. However, direct dynamic assessment of the fundamental partial reactions of substrate binding and activity has proven 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 synchronously with monitoring proteolytic degradation of the substrate. SM recordings enabled us to determine how the overall activity of Savinase and two mutants relies on binding efficiency, enzymatic turnover and activity per binding event. Analysis of residence times revealed three characteristic binding states. Mutations were found to dominantly alter the likelihood of sampling the long lived state, with lifetimes longer than 30 seconds, indicating this state contributes to overall activity and supporting a level of processivity for Savinase. This observation challenges conventional expectations, as the protease has no characterized substrate binding site, or binding domain, aside from the active site. These insights, inaccessible through conventional assays, offer new perspectives for engineering proteases with improved hydrolytic performance.