Design, Synthesis and Evaluation of Specific Substrates for E. coli Penicillin-Binding Protein 2

synthesis. All members of this family of enzymes are susceptible to inhibition by ?-lactam antibiotics, and are consequently referred to as penicillin-binding proteins (PBPs). Unique amongst this family of enzymes in E. coli is PBP2, which is the only enzyme that is susceptible to inhibition by the amidinopenicillin mecillinam. Intriguingly, mecillinam is also unique amongst ?-lactams, bearing an unusual, positively charged amidine side chain. Despite attracting significant interest over several decades, the nature of mecillinam's exquisitely monogamous relationship with PBP2 remains obscure. This lab has previously shown that a given peptidoglycan-mimetic side chain confers a similar change in target reactivity to both ?-lactams and peptide substrates. This result would seem to imply that the mecillinam side chain, which dramatically increases the affinity and specificity of penicillin for E. coli PBP2, would therefore also increase the affinity and specificity of peptide and thiodepsipeptide substrates for PBP2. This thesis describes the creation of such compounds and their kinetic evaluation with E. coli PBP2. The results of these experiments suggest that, unlike the effect of the peptidoglycan-mimetic side chains, the mecillinam side chain confers increased PBP2 affinity only to penicillin and not to substrates. It remains unclear why E. coli PBP2 is able to bind and react with mecillinam, but not with substrates bearing the mecillinam side chain. Sequence alignments of a variety of PBPs reveal that E. coli PBP2 contains an unusual SXD motif in its active site, rather than the SXN motif which is strictly conserved in most other PBPs. It is an intriguing coincidence, indeed, that E. coli PBP2 is unique in both its active site structure and in its reactivity with ?-lactams. Astonishingly, this coincidence appears to have completely escaped discussion until now. This thesis explores each manifestation of the functional importance of the Asn-position of the SXN motif, including its role in substrate recognition, the reactivity of mecillinam with ?-lactamases, the kinetic properties of a variety of SXN* mutants, the identification of bacterial species with SXD-containing PBPs, and molecular dynamics simulations of a homology model of E. coli PBP2. The information compiled and examined in this thesis supports the hypothesis that Asp 132 of the SXD motif of E. coli PBP2 is solely responsible for the enzyme's unusual behavior, and that an electrostatic interaction between the positively charged amidine and the negatively charged Asp is the source of mecillinam's specificity for E. coli PBP2. This proposition suggests promising new directions for further characterization of this elusive enzyme.