Why can higher thermostability reduce catalytic efficiency?

Why

Catalysis often requires conformational sampling for substrate entry, active-site rearrangement or product release. A mutation that locks a mobile region may increase melting temperature while lowering turnover or altering Km. Yet many stabilizing changes occur away from the active site and may preserve or even improve catalysis.

Measure parent and variant with purified active protein at multiple temperatures using initial-rate kinetics. Determine kcat, Km and kcat/Km rather than relying on one substrate concentration. Also test thermal inactivation over time: a thermostable enzyme with slightly lower instantaneous activity may produce more product over an extended industrial cycle.

For example, reduced activity at 25 degrees Celsius tells little about productivity at 55 degrees Celsius without thermal-lifetime data. Check that altered expression, aggregation or cofactor occupancy is not masquerading as a kinetic change. The engineering target is the actual use-case objective, not maximization of Tm alone.