Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein engineering

Background:

• The function of amino acid residues distant from an enzyme's active site is not fully understood.
• These distal residues may play a role in the complete enzyme catalytic cycle.

Purpose of the Study:

• To investigate how mutations in distal amino acid residues affect enzyme catalysis.
• To compare the effects of active-site versus distal mutations on enzyme function.

Main Methods:

• Engineering of mutant de novo Kemp eliminases with active-site or distal mutations.
• Kinetic analyses to measure enzyme activity.
• X-ray crystallography and molecular dynamics simulations to study enzyme structure and dynamics.

Main Results:

• Active-site mutations enhance chemical transformation by creating preorganized catalytic sites.
• Distal mutations improve substrate binding and product release by altering enzyme dynamics and active-site accessibility.
• Synergistic effects between active-site and distal mutations lead to improved overall enzyme activity.

Conclusions:

• Enzyme active sites are necessary but not sufficient for optimal catalysis.
• Distal residues play critical roles in enhancing enzyme efficiency by modulating structural dynamics.
• These findings provide valuable insights for enzyme design and engineering.