ALTERING ENZYMATIC-ACTIVITY - RECRUITMENT OF CARBOXYPEPTIDASE ACTIVITY INTO AN RTEM BETA-LACTAMASE PENICILLIN-BINDING PROTEIN-5 CHIMERA

The D-Ala-D-Ala carboxypeptidases/transpeptidases (penicillin-binding proteins, PBPs) share considerable structural homology with class A β-lactamases (EC 3.5.2.6), although these β-lactamases have no observable D-Ala-D-Ala carboxypeptidase activity. With the objective of recruiting such activity into a β-lactamase background, we have prepared a chimeric protein by inserting a 28-amino acid segment of PBP-5 of Escherichia coli in place of the corresponding region of the RTEM-1 β-lactamase. The segment thus inserted encompasses two residues conserved in both families: Ser-70, which forms the acyl-enzyme intermediate during β-lactam hydrolysis, and Lys-73, whose presence has been shown to be necessary for catalysis. This chimera involves changes of 18 residues and gives a protein that differs at 7% of the residues from the parent. Whereas RTEM β-lactamase has no D-Ala-D-Ala carboxypeptidase activity, that of the chimera is significant and is, in fact, about 1% the activity of PBP-5 on diacetyl-L-Lys-D-Ala-D-Ala; in terms of free energy of activation, the chimera stabilizes the transition state for the reaction to within about 2.7 kcal/mol of the stabilization achieved by PBP-5. Furthermore, the chimera catalyzes hydrolysis exclusively at the carboxyl-terminal amide bond which is the site of cleavage by D-Ala-D-Ala carboxypeptidase. Though containing all those residues that are conserved throughout class A β-lactamases and are thought to be essential for β-lactamase activity, the chimera has considerably reduced activity (~ 10-5) on penams such as penicillins and ampicillins as substrates. As a catalyst, the chimera shows an induction period of ~ 30 min, reflecting a slow conformational rearrangement from an inactive precursor to the active enzyme.