Re-engineering of CYP2C9 to probe acid-base substrate selectivity

Area of Science:

• Biochemistry
• Enzymology
• Drug Metabolism

Background:

• Cytochrome P450 2C9 (CYP2C9) enzymes commonly bind weakly acidic ligands.
• Crystallography reveals this interaction involves a charge-pairing between anionic substrates and active site residue R108.

Purpose of the Study:

• To re-engineer CYP2C9 to better accommodate basic ligands.
• Investigate the role of active site residue R108 in substrate selectivity.

Main Methods:

• Site-directed mutagenesis to create R108E and R108E/D293N CYP2C9 mutants.
• Expression and purification of native and mutant CYP2C9 enzymes.
• Enzyme kinetics and spectral analysis using various ligands: (S)-warfarin, diclofenac, pyrene, propranolol, and ibuprofen amine.

Main Results:

• The R108E mutant retained pyrene 1-hydroxylation activity but lost activity with diclofenac and (S)-warfarin.
• The R108E/D293N double mutant showed reduced selectivity for control ligands.
• Neither mutant enhanced propranolol metabolism, and ibuprofen amine binding weakened with mutations.

Conclusions:

• Residue 108 is critical for CYP2C9's acidic substrate selectivity.
• The CYP2C9 active site is adaptable but re-engineering for different ligand types is complex.
• Mutagenesis studies provide insights into enzyme-substrate interactions and active site plasticity.