On the variation of structural divergence among residues in enzyme evolution

Structural divergence varies among protein residues, yet this variation has been largely overlooked compared with the well-studied case of sequence rate variation. Here we show that, in families of functionally conserved homologous enzymes, structural divergence increases with both residue flexibility and distance from the active site. Although these properties are correlated, modelling reveals that the pattern arises from two independent types of evolutionary constraints: non-functional and functional. The balance between these constraints varies widely across enzyme families, from non-functional to functional dominance. As functional constraints strengthen, structural divergence patterns are reshaped, becoming increasingly distinct from flexibility patterns and breaking the commonly assumed correspondence between evolutionary and dynamical structural ensembles. Active sites are more structurally conserved than average, but this conservation stems not only from functional constraints. Because active sites typically lie in rigid regions where non-functional constraints are high, both constraint types contribute comparably on average, with dominance shifting from one to the other depending on active-site rigidity. Together, these findings revise two long-standing assumptions: that evolutionary structural variation universally mirrors protein dynamics, and that active-site conservation reflects functional requirements alone. Both depend on the balance between non-functional and functional constraints that shape enzyme structural evolution.