Why can a stabilizing mutation reduce protein activity?

Why

Protein stability describes energetics and persistence of folded states, whereas a useful enzyme must bind substrate and move through catalytic conformations. A mutation near a flexible loop may raise its melting temperature yet restrict motion required to release product or align catalytic residues. It may also change substrate affinity, pH dependence or expression.

Compare purified parent and variant at the same active-enzyme concentration using thermal stability, aggregation, steady-state kinetics and relevant operating conditions. For example, a variant could retain activity longer at 60 degrees Celsius but have a lower room-temperature kcat. That might still be a better industrial enzyme when total product over a long run is the objective.

A higher Tm alone does not demonstrate better function and does not always predict storage stability. Interpret trade-offs in context and seek combinations of variants that improve multiple properties rather than assuming rigidity must always reduce catalysis.