Relocation of the catalytic carboxylate group in class A beta-lactamase: the structure and function of the mutant enzyme Glu166 -> Gln : Asn170 -> Asp

The hydrolysis of beta-lactam antibiotics by the serine-beta-lactamases pro ceeds via an acyl-enzyme intermediate. In the class A enzymes, a key cataly tic residue, Glu166, activates a water molecule for nucleophilic attack on the acyl-enzyme intermediate. The active site architecture raises the possi bility that the location of the catalytic carboxylate group may be shifted while still maintaining close proximity to the hydrolytic water molecule, A double mutant of the Staphylococcus aureus PC1 beta-lactamase, E166Q:N170D , was produced, with the carboxylate group shifted to position 170 of the p olypeptide chain. A mutant protein, E166Q, without a carboxylate group and with abolished deacylation, was produced as a control. The kinetics of the two mutant proteins have been analyzed and the crystal structure of the dou ble mutant protein has been determined. The kinetic data confirmed that dea cylation was restored in E166Q:N170D beta-lactamase, albeit not to the leve l of the wild-type enzyme. In addition, the kinetics of the double mutant e nzyme follows progressive inactivation, characterized by initial fast rates and final slower rates. The addition of ammonium sulfate increases the siz e of the initial burst, consistent with stabilization of the active form of the enzyme by salt. The crystal structure reveals that the overall fold of the E166Q:N170D enzyme is similar to that of native beta-lactamase. Howeve r, high crystallographic temperature factors are associated with the Omega- loop region and some of the side chains, including Asp170, are partially or completely disordered, The structure provides a rationale for the progress ive inactivation of the Asp170-containing mutant, suggesting that the flexi ble Omega-loop may be readily perturbed by the substrate such that Asp170's carboxylate group is not always poised to facilitate hydrolysis.