Deacylation Mechanism and Kinetics of Acyl–Enzyme Complex of Class C β‑Lactamase and Cephalothin

Understanding the molecular details
of antibiotic resistance by
the bacterial enzymes β-lactamases is vital for the development
of novel antibiotics and inhibitors. In this spirit, the detailed
mechanism of deacylation of the acyl–enzyme complex formed
by cephalothin and class C β-lactamase is investigated here
using hybrid quantum-mechanical/molecular-mechanical molecular dynamics
methods. The roles of various active-site residues and substrate in
the deacylation reaction are elucidated. We identify the base that
activates the hydrolyzing water molecule and the residue that protonates
the catalytic serine (Ser64). Conformational changes in
the active sites and proton transfers that potentiate the efficiency
of the deacylation reaction are presented. We have also characterized
the oxyanion holes and other H-bonding interactions that stabilize
the reaction intermediates. Together with the kinetic and mechanistic
details of the acylation reaction, we analyze the complete mechanism
and the overall kinetics of the drug hydrolysis. Finally, the apparent
rate-determining step in the drug hydrolysis is scrutinized.