Protonation states of active-site lysines of penicillin-binding protein 6 from Escherichia coli and the mechanistic implications.

The protonation states of the two active-site lysines (Lys69 and Lys235) of PBP 6 of Escherichia coli were explored to understand the active site chemistry of this enzyme. Each lysine was individually mutated to cysteine, and the resultant two mutant proteins were purified to homogeneity. Each protein was denatured, and its cysteine was chemically modified to produce an S-aminoethylated cysteine (γ-thialysine) residue. Following renaturation, the evaluation of the kinetics of the dd-carboxypeptidase activity of PBP 6 as a function of pH was found consistent with one lysine in its free-base (Lys69) and the other in the protonated state (Lys235) for optimal catalysis. The experimental estimates for their pKa values were compared with the pKa values calculated computationally, using molecular-dynamics simulations and a thermodynamic cycle. Study of the γ-thialysine69 showed that lysine at position 69 influenced the basic limb of catalysis, consistent with the fact that the two lysine side chains are in proximity to each other in the active site. Based on these observations, a reaction sequence for PBP 6 is proposed, wherein protonated Lys235 serves as the electrostatic substrate anchor and Lys69 as the conduit for protons in the course of the acylation and deacylation half-reactions.