Single-molecule studies reveal method for tuning the heterogeneous activity of alkaline phosphatase

Single-molecule-enzymology (SME) methods have enabled the observation of heterogeneous catalytic activities within a single enzyme population. Heterogeneous activity is hypothesized to originate from conformational changes in the enzyme that result from changes in the local environment leading to catalytically active substates. Here, we use SME to investigate the mechanisms of heterogeneous activity exhibited by tissue nonspecific alkaline phosphatase (TNSALP), which reveals two subpopulations with different catalytic activities. We show the effect of pH and temperature on the distribution of TNSALP activity and confirm the presence of two subpopulations attributed to half- and fully active TNSALP substates. We provide mechanistic insight about protein structure using molecular dynamic simulations and show pH- and temperature-dependent conformational transitions that corroborate experimentally observed changes in TNSALP activity. These results show the utility of SME to understand heterogeneous enzyme activity and demonstrate a simple approach using pH and temperature to tune catalytic activity within an enzyme population.

Automated summary

Single-molecule enzymology methods allow for the study of heterogeneous catalytic activities within enzyme populations. This study explores the mechanisms of heterogeneous activity in tissue nonspecific alkaline phosphatase (TNSALP), revealing two subpopulations with different catalytic activities. pH and temperature are shown to influence TNSALP activity, and molecular dynamics simulations provide insight into conformational transitions that align with experimental observations. These findings demonstrate the usefulness of single-molecule enzymology in understanding heterogeneous enzyme activity and provide a simple approach for tuning catalytic activity within enzyme populations using pH and temperature.