Conserved Asp233–Asp246 hydrogen bond modulates active site dynamics in class A β‐lactamases

Abstract

Rigid enzymes catalyze chemical reactions by stabilizing the transition state through a specific conformation. Catalytic residues are precisely positioned, and enzyme dynamics are kept to a minimum. It is thought that conserved residues around the active site (second‐shell residues) play a crucial role in positioning catalytic residues. Asp233 and Asp246 are two highly conserved second‐shell residues in class A β‐lactamases, rigid enzymes that inactivate β‐lactam antibiotics. The two aspartates share a short hydrogen bond, linking β‐strands 3 and 4. The role of this interaction in Mycobacterium tuberculosis β‐lactamase BlaC was studied by mutating the Asp residues to Ala. Disruption of the hydrogen bond subtly affects the activity and stability of the enzyme, suggesting the interaction helps to fine‐tune the active site. The effects are larger for BlaC D246A than for D233A, indicating that effects cannot solely be attributed to the loss of the hydrogen bond. Molecular dynamics calculations indicate a shift in the conformational landscape due to the mutations, altering the conformational equilibria of the catalytic residues toward less active states. The results illustrate that second‐shell residues act as a complex network that supports the efficient positioning of the catalytic residues.