Remarkable activity enhancement of thermolysin mutants

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Enzyme modification via amino acid substitution often decreases biological activity.
• Natural enzymes are generally considered evolutionarily optimized for maximal activity.
• Thermolysin (EC.3.4.24.4) is a well-characterized metalloendopeptidase.

Purpose of the Study:

• To investigate amino acid substitutions at a specific site in thermolysin.
• To identify factors influencing enzyme activity and stability.
• To challenge the notion of universal evolutionary optimization for enzyme activity.

Main Methods:

• Site-directed mutagenesis of thermolysin.
• Assays to measure hydrolytic activity.
• Thermal stability measurements.

Main Results:

• Amino acid replacement at a key site in thermolysin significantly increased hydrolytic activity.
• A negative correlation was observed between enzyme activity and thermal stability.
• Increased flexibility around the substrate binding site correlated with higher activity.

Conclusions:

• A specific site in thermolysin allows for activity enhancement through amino acid substitution.
• Evolution may have prioritized thermal stability over maximal activity at this site.
• Molecular flexibility is a critical factor in the observed activity-stability trade-off.