Crystal Structure of 6α-(Hydroxymethyl)penicillanate Complexed to the TEM-1 β-Lactamase from <i>Escherichia coli</i>: Evidence on the Mechanism of Action of a Novel Inhibitor Designed by a Computer-Aided Process

Abstract

The crystal structure of the complex of the TEM-1 β-lactamase from Escherichia coli inhibited by 6α-(hydroxymethyl)penicillanic acid (1) is reported herein. This is the first structure for an acyl-enzyme intermediate with a substrate reported for a native class A β-lactamase. This compound was designed and synthesized as a molecule that would acylate the active site of the enzyme, but would resist deacylation by virtue of the fact that its C6α hydroxymethyl moiety was expected to occupy the space near the hydrolytic water molecule (J. Am. Chem. Soc. 1995, 117, 11055). The crystal structure of the acyl-enzyme species is closely similar to one of the two energy-minimized acyl-enzyme models generated in the course of the design aspect of the work. The crystal structure provides evidence for a number of mechanistic features for the inhibition process and the ultimate recovery of the activity. Our results reported herein are consistent with the side-chain carboxylate of Glu-166 being the active-site basic function that activates the hydrolytic water for the deacylation step in the course of catalysis by class A β-lactamases. The design principles applied for compound 1 hold the promise of general utility for development of novel inhibitors for other hydrolytic enzymes.