Insights into the molecular basis for the carbenicillinase activity of PSE-4 beta-lactamase from crystallographic and kinetic studies

PSE-4 is a class A beta -lactamase produced by strains of Pseudomonas aerug inosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild-type PSE-4 carbenicillinase has been determi ned to 1.95 Angstrom resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. A superposition o f the PSE-4 structure with that of TEM-1 shows a rms deviation of 1.3 Angst rom for 263 C alpha atoms. Most carbenicillinases are unique among class A beta -lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kine tic characterization of a R234K PSE-4 mutant reveals a 50-fold reduction in k(cat)/K-m and confirms the importance of Arg 234 for carbenicillinase act ivity. A comparison of the structure of the R234K mutant refined to 1.75 An gstrom resolution with the wild-type structure shows that Arg 234 stabilize s an alternate conformation of the Ser 130 side chain, not seen in other cl ass A beta -lactamase structures. Our molecular modeling studies suggest th at the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the ca rbenicillin alpha -carboxylate group and the conserved side chain of Asn 17 0. The alternate conformation of the catalytic Ser 130 in wild-type PSE-4 m ay be involved in accommodating this shift in the bound substrate position.