Contributions of aspartate 49 and phenylalanine 142 residues of a tight binding inhibitory protein of beta-lactamases

beta-Lactamases are bacterial enzymes that hydrolyze beta-lactam antibiotic s to render them inactive. The beta-lactamase inhibitor protein (BLIP) of S treptomyces clavuligerus, is a potent inhibitor of several beta-lactamases, including the TEM-1 enzyme (K-i = 0.6 nM). Evidence from the TEM-1/BLIP co -crystal suggests that two BLIP residues, Asp-49 and Phe-142, mimic interac tions made by penicillin G when bound in the active site of TEM-1. To deter mine the importance of these two residues, a heterologous expression system for BLIP was established in Escherichia coli. Site-directed mutagenesis wa s used to change Asp-49 and Phe-142 to alanine, and inhibition constants (K -i) for both mutants were determined. Each mutation increases the K-i for B LIP inhibition of TEM-1 beta-lactamase approximately 100-fold To address ho w these two positions effect the specificity of beta-lactamase binding, K-i values were determined for the interaction of wild-type BLIP, as well as t he D49A and F142A mutants, with two extended spectrum beta-lactamases (the G238S and the E104K TEM variants). Positions 104 and 238 are located in the BLIP/beta-lactamase interface. Interestingly, the three BLIP proteins inhi bited the G238S beta-lactamase mutant to the same degree that they inhibite d TEM-1, However, wild-type BLIP has a higher K-i for the E104K beta-lactam ase mutant, suggesting that interactions between BLIP and beta-lactamase re sidue Glu-104 are important for wild-type levels of BLIP inhibition.