Directed Evolution of Protoglobin Optimizes the Enzyme Electric Field

To unravel why computational design
fails in creating viable enzymes,
while directed evolution (DE) succeeds, our research delves into the
laboratory evolution of protoglobin. DE has adapted this protein to
efficiently catalyze carbene transfer reactions. We show that the
previously proposed enhanced substrate access and binding alone cannot
account for increased yields during DE. The 3D electric field in the
entire active site is tracked through protein dynamics, clustered
using the affinity propagation algorithm, and subjected to principal
component analysis. This analysis reveals notable changes in the electric
field with DE, where distinct field topologies influence transition
state energetics and mechanism. A chemically meaningful field component
emerges and takes the lead during DE and facilitates crossing the
barrier to carbene transfer. Our findings underscore intrinsic electric
field dynamic’s influence on enzyme function, the ability of
the field to switch mechanisms within the same protein, and the crucial
role of the field in enzyme design.