Design of a switchable eliminase.

Area of Science:

• Biochemistry and Molecular Biology
• Protein Engineering
• Enzyme Catalysis

Background:

• Enzyme active sites utilize tuned side-chain properties, including enhanced basicity of carboxylates via dehydration, to achieve catalysis.
• Allosteric regulation in enzymes leverages ligand binding to stabilize catalytically active protein conformations.
• Protein folding and ligand binding provide free energy to tune functional groups for specific chemical properties.

Purpose of the Study:

• To demonstrate the interplay of protein folding energetics and functional group tuning in enzyme design.
• To convert calmodulin (CaM), a calcium-binding protein, into an allosterically controlled enzyme (eliminase).
• To create a minimal, computationally designed enzyme catalyst.

Main Methods:

• Computational identification of a calmodulin mutant enabling carboxylate accommodation as a general base.
• Engineering a mutant to interact productively in the Michaelis complex and stabilize the transition state.
• Characterization of the engineered enzyme (AlleyCat) using pH-rate profiles and site-directed mutagenesis.

Main Results:

• A single mutation in calmodulin created AlleyCat, an allosterically regulated enzyme catalyzing the Kemp elimination reaction.
• AlleyCat exhibited a pH-rate profile consistent with its engineered active site and was inactivated by active site mutation.
• The catalytic activity of this minimal 75-residue enzyme rivaled that of more complex, computationally redesigned enzymes.

Conclusions:

• Protein folding energetics and functional group tuning can be harnessed to engineer novel enzymatic activity.
• Calmodulin can be repurposed into a minimal, allosterically regulated enzyme through targeted mutation.
• This study highlights a powerful strategy for designing simple, efficient enzyme catalysts.