Site-saturation Mutagenesis of Tyr-105 Reveals Its Importance in Substrate Stabilization and Discrimination in TEM-1 β-Lactamase
The conserved Class A β-lactamase active site residue Tyr-105 was substituted by saturation mutagenesis in TEM-1 β-lactamase fromEscherichia coliin order to clarify its role in enzyme activity and in substrate stabilization and discrimination. Minimum inhibitory concentrations were calculated forE. colicells harboring each Y105Xmutant in the presence of various penicillin and cephalosporin antibiotics. We found that only aromatic residues as well as asparagine replacements conferred highin vivosurvival rates for all substrates tested. At position 105, the small residues alanine and glycine provide weak substrate discrimination as evidenced by the difference in benzylpenicillin hydrolysis relative to cephalothin, two typical penicillin and cephalosporin antibiotics. Kinetic analyses of mutants of interest revealed that the Y105Xreplacements have a greater effect onKmthankcat, highlighting the importance of Tyr-105 in substrate recognition. Finally, by performing a short molecular dynamics study on a restricted set of Y105Xmutants of TEM-1, we found that the strong aromatic bias observed at position 105 in Class A β-lactamases is primarily defined by a structural requirement, selecting planar residues that form a stabilizing wall to the active site. The adopted conformation of residue 105 prevents detrimental steric interactions with the substrate molecule in the active site cavity and provides a rationalization for the strong aromatic bias found in nature at this position among Class A β-lactamases.