A theoretical study on the substrate deacylation mechanism of class C beta-lactamase.

The whole reaction of the deacylation of class C beta-lactamase was investigated by performing quantum chemical calculations under physiological conditions. In this study, the X-ray crystallographic structure of the inhibitor moxalactam-bound class C beta-lactamase (Patera et al. J. Am. Chem. Soc. 2000, 122, 10504-10512.) was utilized and moxalactam was changed into the substrate cefaclor. A model for quantum chemical calculations was constructed using an energy-minimized structure of the substrate-bound enzyme obtained by molecular mechanics calculation, in which the enzyme was soaked in thousands of TIP3P water molecules. It was found that the deacylation reaction consisted of two elementary processes. The first process was formation of a tetrahedral intermediate, which was initiated by the activation of catalytic water by Tyr150, and the second process was detachment of the hydroxylated substrate from the enzyme, which associated with proton transfer from the side chain of Lys67 to Ser64O(gamma). The first process is a rate-determining process, and the activation energy was estimated to be 30.47 kcal/mol from density functional theory calculations considering electron correlation (B3LYP/6-31G**). The side chain of Tyr150 was initially in a deprotonated state and was stably present in the active site of the acyl-enzyme complex, being held by Lys67 and Lys315 cooperatively.