Exploring the Mechanism of β‑Lactam Ring Protonation in the Class A β‑lactamase Acylation Mechanism Using Neutron and X‑ray Crystallography

The
catalytic mechanism of class A β-lactamases is often
debated due in part to the large number of amino acids that interact
with bound β-lactam substrates. The role and function of the
conserved residue Lys 73 in the catalytic mechanism of class A type
β-lactamase enzymes is still not well understood after decades
of scientific research. To better elucidate the functions of this
vital residue, we used both neutron and high-resolution X-ray diffraction
to examine both the structures of the ligand free protein and the
acyl–enzyme complex of perdeuterated E166A Toho-1 β-lactamase
with the antibiotic cefotaxime. The E166A mutant lacks a critical
glutamate residue that has a key role in the deacylation step of the
catalytic mechanism, allowing the acyl–enzyme adduct to be
captured for study. In our ligand free structures, Lys 73 is present
in a single conformation, however in all of our acyl–enzyme
structures, Lys 73 is present in two different conformations, in which
one conformer is closer to Ser 70 while the other conformer is positioned
closer to Ser 130, which supports the existence of a possible pathway
by which proton transfer from Lys 73 to Ser 130 can occur. This and
further clarifications of the role of Lys 73 in the acylation mechanism
may facilitate the design of inhibitors that capitalize on the enzyme’s
native machinery.