pKa calculations for class A beta-lactamases: methodological and mechanistic implications

Area of Science:

• Biochemistry
• Enzymology
• Computational Biology

Background:

• Beta-lactamases confer resistance to beta-lactam antibiotics by hydrolyzing them.
• The precise catalytic mechanism, particularly the general base in the acylation step, remains debated.
• Lysine 73 (Lys73) has been proposed as a potential general base, based on prior pKa calculations and structural data.

Purpose of the Study:

• To computationally determine the pKa values of titratable residues in TEM-1 and Bacillus licheniformis class A beta-lactamases.
• To evaluate the role of Lys73 as a general base in the enzyme's acylation mechanism.
• To investigate the proton transfer pathway involving Serine 70 (Ser70) and Glutamate 166 (Glu166).

Main Methods:

• Utilized a continuum electrostatic model with advanced treatment for multiple titration sites.
• Calculated pKa values for all titratable residues in substrate-free TEM-1 and B. licheniformis beta-lactamases.
• Performed computational analysis, including mutation of Glu166, to assess Lys73's proton abstraction capability.

Main Results:

• Computed pKa for Lys73 in both enzymes was significantly above 10, aligning with experimental data for TEM-1.
• The calculated pKa of Lys73 is inconsistent with its proposed role as a general base.
• Mutation of Glu166 did not sufficiently lower Lys73's pKa to support its function as a proton abstractor.

Conclusions:

• Lys73 is unlikely to function as the general base in the acylation step of TEM-1 and B. licheniformis beta-lactamases.
• Results strongly support a mechanism involving proton transfer from the active site Ser70 to the carboxylate of Glu166.
• This study refines our understanding of beta-lactamase catalytic mechanisms and antibiotic resistance.