Transition Path Sampling Study of the Feruloyl Esterase Mechanism

Area of Science:

• Biochemistry
• Enzymology
• Computational Chemistry

Background:

• Serine hydrolases are vital enzymes catalyzing peptide and ester bond cleavage, with broad applications and therapeutic potential.
• Their catalytic mechanism involves a serine-histidine-aspartate/glutamate triad, but the proton shuttle mechanism of histidine remains debated.

Purpose of the Study:

• To elucidate the detailed reaction mechanism of serine hydrolases using a model enzyme.
• To investigate the role of active site dynamics in the catalytic efficiency of serine hydrolases.

Main Methods:

• Employed quantum mechanics/molecular mechanics (QM/MM)-based transition path sampling.
• Utilized *Aspergillus niger* feruloyl esterase A (AnFaeA) as a model system.
• Calculated reaction coordinates and rate constants.

Main Results:

• Identified a concerted reaction mechanism where the transition state coincides with the tetrahedral intermediate.
• Observed histidine reorientation on a femtosecond timescale, supporting the 'moving histidine' mechanism over the 'ring flip' mechanism.
• Found concerted motions of aspartate stabilizing the transition state and facilitating nucleophilic attack.

Conclusions:

• The study reveals that active site dynamics, specifically histidine reorientation, are pivotal in the serine hydrolase catalytic mechanism.
• The deacylation step is rate-determining for AnFaeA, with a calculated rate constant of 66 s⁻¹.
• The elucidated mechanism provides insights applicable to other serine hydrolases.