8 research outputs found
Substrate Binding Induces Conformational Changes in a Class A β‑lactamase That Prime It for Catalysis
The
emergence and dissemination of bacterial resistance to β-lactam
antibiotics via β-lactamase enzymes is a serious problem in
clinical settings, often leaving few treatment options for infections
resulting from multidrug-resistant superbugs. Understanding the catalytic
mechanism of β-lactamases is important for developing strategies
to overcome resistance. Binding of a substrate in the active site
of an enzyme can alter the conformations and p<i>K</i><sub>a</sub>s of catalytic residues, thereby contributing to enzyme catalysis.
Here we report X-ray and neutron crystal structures of the class A
Toho-1 β-lactamase in the apo form and an X-ray structure of
a Michaelis-like complex with the cephalosporin antibiotic cefotaxime
in the active site. Comparison of these structures reveals that substrate
binding induces a series of changes. The side chains of conserved
residues important in catalysis, Lys73 and Tyr105, and the main chain
of Ser130 alter their conformations, with Nζ of Lys73 moving
closer to the position of the conserved catalytic nucleophile Ser70.
This movement of Lys73 closer to Ser70 is consistent with proton transfer
between the two residues prior to acylation. In combination with the
tightly bound catalytic water molecule located between Glu166 and
the position of Ser70, the enzyme is primed for catalysis when Ser70
is activated for nucleophilic attack of the β-lactam ring. Quantum
mechanical/molecular mechanical (QM/MM) free energy simulations of
models of the wild-type enzyme show that proton transfer from the
Nζ of Lys73 to the Oε2 atom of Glu166 is more thermodynamically
favorable than when it is absent. Taken together, our findings indicate
that substrate binding enhances the favorability of the initial proton
transfer steps that precede the formation of the acyl-enzyme intermediate