The active site protonation states of perdeuterated Toho-1 ß-lactamase determined by neutron diffraction support a role for Glu166 as the general base in acylation.

ABSTRACT

Room temperature neutron diffraction data of the fully perdeuterated Toho-1 R274N/R276N double mutant ß-lactamase in the apo form were used to visualize deuterium atoms within the active site of the enzyme. This perdeuterated neutron structure of the Toho-1 R274N/R276N reveals the clearest picture yet of the ground-state active site protonation states and the complete hydrogen-bonding network in a ß-lactamase enzyme. The ground-state active site protonation states detailed in this neutron diffraction study are consistent with previous high-resolution X-ray studies that support the role of Glu166 as the general base during the acylation reaction in the class A ß-lactamase reaction pathway.