Single-molecule kinetic exploration of functional sub-states in an evolving phosphotriesterase.

Enzymes can exist in multiple stable functional states or sub-states, revealing substantial molecule-to-molecule heterogeneity in catalytic rates even within populations expressed from a clonal gene. Increasing evidence suggests that biological systems have evolved to exploit these functional sub-states to regulate reaction rates and enzyme multi-functionality or promiscuity. Thus, understanding such heterogeneity in evolutionary dynamics is central to elucidating the mechanisms underlying enzyme adaptability and evolvability. To address this, we analyze the evolutionary changes in functional sub-states using single-molecule kinetics. We measure the functional sub-states of wild-type phosphotriesterase (PTE) and 18 evolved variants spanning a trajectory from native PTE activity to promiscuous arylesterase function. This is further supported by an investigation into the conformational sub-states of selected variants using molecular dynamics simulations. Our results reveal that functional optimization is tightly coupled with the redistribution of underlying functional and conformational sub-states, particularly involving the open and closed dynamics of functionally essential loop 7. These findings provide direct evidence that enzyme evolution involves coordinated shifts in conformational heterogeneity and its functional manifestations, together shaping enzyme function.