Active site studies of beta-lactamases and DD-peptidases: Part 1. Kinetic and thermodynamic evaluation of phosphonates as beta-lactamase inhibitors. Part 2. Peptide substrate specificity studies in DD-peptidases

The class C beta-lactamase of Enterobacter cloacae P99 is irreversibly inactivated by aryl phosphonate monoester monoanions. They react with the active site serine nucleophile in the enzyme to form pentacoordinated transition states/intermediates which rapidly collapse to tetrahedral adducts. The interactions that stabilize these pentacoordinated phosphonate transition states and tetrahedral phosphonate adducts were evaluated against structurally analogous tetrahedral boronate adducts (which are believed to be transition state analogs) by linear free energy correlations. The kinetics of reaction of these inhibitors with the P99 beta-lactamase showed that the pentacoordinated phosphonate transition state is not a good mimic of the tetrahedral boronate adduct formed in the active site of the enzyme. The active site functional groups must interact in a qualitatively different fashion with these differently coordinated species. Studies of the thermodynamics of interaction of the aryl phosphonates with the P99 beta-lactamase, showed that the degree of similarity of the tetracoordinated phosphonate derivatives of the enzyme to acyl transfer transition states cannot generally be assessed by a simple thermodynamic cycle.; Glycyl-L-alpha-amino-epsilon-pimelyl-D-alanyl-D-alanine (GPA) is a very specific substrate for the Streptomyces R61 DD-peptidase (kcat = 69 s-1; Km = 7.9 muM; kcat/Km = 8.7 x 106 M-1 s-1). Since the high specificity was thought to originate from the glycyl-L-alpha-amino-epsilon-pimelyl side chain in GPA, peptide substrates devoid of the specificity elements of this moiety were synthesized and evaluated against the enzyme. A major difficulty in the synthesis of the peptide substrates was largely overcome by a disconnection strategy where a sulfur was introduced in the pimelyl side chain of GPA. The kinetics of reaction of these substrates with the R61 DD-peptidase showed that any modification in the side chain severely impairs the carboxypeptidase activity of these substrates towards the enzyme.; The synthetic methodology described above was then extended to prepare substrates for the Actinomadura R39 DD-peptidase and E. coli PBP2 & PBP5. The Actinomadura R39 DD-peptidase shows less specificity for the side chain than the R61 enzyme, whereas E. coli PBP2 and PBP5 do not catalyze any peptidase reaction of these substrates in vitro. Finally, an inhibitor based on the side chain specificity was designed, synthesized and evaluated against the R61 DD-peptidase.