Single-Molecule Kinetic Exploration of Functional Substates in an Evolving Phosphotriesterase

Enzymes achieve catalysis by dynamically sampling diverse conformational states. Beyond this plasticity, individual enzyme molecules occupy metastable substates, forming an ensemble of functional substates within a population. Since shifts in functional substate dynamics drive phenotypic variation, their evolutionary trajectories are central to the emergence of new functions. However, the challenge of measuring functional substates has hindered our understanding of their role in enzyme evolution and the optimization of conformational substates. Here, we address this gap by investigating how functional and conformational substates were modulated during enzyme functional transitions, using single-molecule kinetic assays and molecular dynamics simulations. We analyzed wild-type phosphotriesterase (PTE) and 18 evolved variants that transitioned from the native PTE to promiscuous arylesterase (AE) activity. Our findings reveal that evolutionary transitions reshape functional and conformational substate landscapes: PTE-specialized variants exhibit broader substate distributions, whereas AE-specialized variants display more uniform substates. These results provide the first direct evidence that enzyme evolution is accompanied by coordinated shifts in functional and conformational substate equilibria, optimizing both for the enzymes catalytic efficiency. This work highlights the power of single-molecule techniques in uncovering how heterogeneous enzyme populations navigate substate transitions and, ultimately, how these transitions shape enzyme evolvability.