Crystal Structure of 6α-(Hydroxymethyl)penicillanate Complexed to the TEM-1 β-Lactamase from Escherichia coli:  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 6R-(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 C 6R 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 energyminimized 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.We reported recently on the properties of 6R-(hydroxymethyl)penicillanic acid (1) as a novel inhibitor for the class A TEM-1 β-lactamase, which was shown to acylate the enzyme active site, but resisted deacylation. 1 This molecule was designed on the basis of the knowledge of the topology of the enzyme active site and the mechanism of enzyme action. The design process commenced with the energy-minimized structure for the acyl-enzyme intermediate for penicillanic acid (2), a reasonably good substrate for the TEM-1 β-lactamase. The 6Rhydroxymethyl group was incorporated into the structure of penicillanate with the intention of displacing the hydrolytic water (Wat-301; referred to as Wat-712 in our previous publication 1 ) from the active site upon enzyme acylation by the substrate. Compound 1 proved to be a potent inhibitor for the TEM-1 β-lactamase. The kinetic parameters for both enzyme inhibition and the enzymic turnover of 1 have already been reported. 1 An interesting aspect of the enzyme inhibition profile was the biphasic nature for both the onset of inhibition and recovery of activity from it. This biphasic character of the kinetics was shown not to be associated with a protein conformational change. 1

Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D β-Lactamase of Acinetobacter baumannii

“…Incidentally, the 6␣HMP rotamer seen in chains A, C, and D of OXA-58 is similar to that seen in 6␣HMP bound to TEM-1 (PDB code 1TEM) (see Fig. S3 in the supplemental material) (33). In the TEM-1-6␣HMP complex, it was noted that the hydroxyl group of 6␣HMP displaced the structurally conserved deacylating water molecule, and that hydrogen bonding with Glu-166 may prevent the glutamate from activating an incoming water molecule.…”

Hydrolytic Mechanism of OXA-58 Enzyme, a Carbapenem-hydrolyzing Class D β-Lactamase from Acinetobacter baumannii

“…The position of this water molecule is crystallographically conserved (31,36,41) unlike in class D enzymes (42)(43)(44). Perturbation of the hydrogen bonding network of this water molecule or the physical displacements of the water molecule by the steric bulk of the C-6 side chains of a ␤-lactam are possible mechanisms to render the ␤-lactamase deacylation-deficient (20,(45)(46)(47). This is the goal in designing new generations of ␤-lactams that are stable toward ␤-lactamases (47).…”

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

“…39,40) Several structural studies on the acylenzyme intermediate analogues indicated that Ser130 made a hydrogen bond with the carboxyl group. 7,[41][42][43] From these experimental findings, the function of Ser130 was thought to be binding a substrate. Díaz et al suggested that Ser130 did not only take part in substrate binding but also enzymatic catalysis.…”