Aromatic stacking interactions govern catalysis in aryl-alcohol oxidase.
Patricia Ferreira1, Aitor Hernández-Ortega2, Fátima Lucas3
1Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, and Instituto de Biocomputación y Física de Sistemas Complejos, Zaragoza, Spain.
The FEBS Journal
|February 3, 2015
Summary
Aryl-alcohol oxidase (AAO) uses Tyr92 for substrate binding, influencing its reaction mechanism. Substituent effects on benzyl alcohols dictate whether AAO follows a ping-pong or ternary complex mechanism.
Area of Science:
- Biochemistry
- Enzymology
- Protein-ligand interactions
Background:
- Aryl-alcohol oxidase (AAO) is crucial for lignin degradation, producing H2O2 by oxidizing primary alcohols.
- The enzyme's catalytic mechanism involves interactions between the Tyr92 residue and alcohol substrates.
Purpose of the Study:
- To elucidate the role of Tyr92 in the catalytic mechanism of Pleurotus eryngii AAO.
- To investigate how substrate substituents influence the reaction kinetics and mechanism.
Main Methods:
- Ligand diffusion studies to analyze substrate-enzyme interactions.
- Bi-substrate kinetics analysis with various substituted benzyl alcohols.
- Site-directed mutagenesis of Tyr92 (to phenylalanine and tryptophan).
- Quantum mechanics calculations of stacking energies.
Main Results:
- Electron-withdrawing substituents (halogens) favor a ping-pong mechanism, correlated with lower stacking energies.
- Electron-donating substituents (methoxy groups) lead to a ternary complex mechanism, associated with higher stacking energies.
- Mutating Tyr92 to phenylalanine had minimal effect, while mutation to tryptophan significantly reduced substrate affinity and catalytic efficiency.
Conclusions:
- Tyr92 plays a critical role in substrate binding and recognition within the AAO active site.
- The nature of substituents on the alcohol substrate dictates the enzyme's kinetic mechanism via modulation of Tyr92 interactions.
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