Understanding the molecular determinants of substrate and inhibitor specificities in the Carbapenemase KPC-2: exploring the roles of Arg220 and Glu276

Abstract

β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A β-lactamase that inactivates carbapenems and β-lactamase inhibitors. Our study reveals that substitutions at R220 of KPC-2 selectively impact catalytic activity toward substrates (50% or greater reduction in k(cat)/K(m)). In addition, we find that residue 220 is central to the mechanism of β-lactamase inhibition/inactivation. Among the variants tested at Ambler position 220, the R220K enzyme is relatively "inhibitor susceptible" (K(i) of 14 ± 1 μM for clavulanic acid versus K(i) of 25 ± 2 μM for KPC-2). Specifically, the R220K enzyme is impaired in its ability to hydrolyze clavulanic acid compared to KPC-2. In contrast, the R220M substitution enzyme demonstrates increased K(m) values for β-lactamase inhibitors (>100 μM for clavulanic acid versus 25 ± 3 μM for the wild type [WT]), which results in inhibitor resistance. Unlike other class A β-lactamases (i.e., SHV-1 and TEM-1), the amino acid present at residue 276 plays a structural rather than kinetic role with substrates or inhibitors. To rationalize these findings, we constructed molecular models of clavulanic acid docked into the active sites of KPC-2 and the "relatively" clavulanic acid-susceptible R220K variant. These models suggest that a major 3.5-Å shift occurs of residue E276 in the R220K variant toward the active S70 site. We anticipate that this shift alters the shape of the active site and the positions of two key water molecules. Modeling also suggests that residue 276 may assist with the positioning of the substrate and inhibitor in the active site. These biochemical and molecular modeling insights bring us one step closer to understanding important structure-activity relationships that define the catalytic and inhibitor-resistant profile of KPC-2 and can assist the design of novel compounds.

Fig 1

Compounds used in this study.

Fig 2

A representation of the KPC-2 β-lactamase (E) reaction pathway with a β-lactamase inhibitor (I) (i.e., clavulanic acid).

Fig 3

Activity curves of the kinetic interactions of clavulanic acid with KPC-2 and the R220M and -K variants. The reaction of 10 nM KPC-2 (A) or the R220M (B) or -K (C) variant with increasing concentrations of clavulanic acid over time was monitored at an absorbance of 482 nm using 50 μM nitrocefin as a reporter substrate for β-lactamase activity. Compared to KPC-2 and the R220M variant, the R220K variant is readily inactivated by clavulanic acid.

Fig 4

Monitoring hydrolysis using clavulanic acid with KPC-2 and the R220K and -M variants. Clavulanic acid was incubated with KPC-2 or the R220M or -K variant, and hydrolysis at 235 nm was monitored over time.

Fig 5

Timed mass spectrometry of the R220K variant with clavulanic acid. In a timed mass spectrometry experiment, clavulanic acid was reacted with the R220K variant. Samples were collected at 1 min, 2.5 min, 5 min, 10 min, and 20 min. An enzyme-alone control was also run (i.e., the R220K variant; 28,118 ± 3 atomic mass units [amu]). Mass adducts of +51 ± 3 amu (propynyl enzyme or enol ether), +69 ± 3 amu (aldehyde), and +88 ± 3 amu (hydrated aldehyde) were observed with the R220K variant.

Fig 6

Molecular modeling of KPC-2 and the R220A, -M, and -K variants. (A) The apo-enzyme of the KPC-2 (gray) was superimposed on the apo-enzymes of the R220A (orange), -M (dark blue), and -K (cyan) variants, revealing a 3.5-Å shift in E276 for the R220K variant. (B) Electrostatic surface of the active site of KPC-2–clavulanic acid (left). The molecular representation of KPC-2 (gray) with clavulanic acid (blue) reveals that Wat1 (red) forms hydrogen bonds (green dashed line) with E166 (top right). The distance (d) in angstroms between E166 and Wat1 throughout the 100-ps simulation (bottom right) is indicated. (C) Electrostatic surface of the active site of the R220K variant-clavulanic acid (left). The molecular representation of the interaction of the R220K variant (gray) with clavulanic acid (blue) indicates that Wat2 (red) is hydrogen bonded (green dashed line) to clavulanic acid and E276 (top right). The distance (d) in angstroms between C7 of clavulanic acid and Wat2 throughout the 5-ps simulation (bottom right) is indicated.

Fig 7

Mechanisms of clavulanic acid interactions with KPC-2 and the R220K variant. (A) Mechanism of clavulanic acid resistance by WT KPC-2. The major pathway is illustrated; only minor amounts of enamine may form and thus are not depicted in this scheme. The S70 nucleophile attacks the β-lactam ring. The deacylation water is positioned close to E166, promoting deacylation of the acyl-enzyme complex. (B) Mechanism of clavulanic acid inhibition of the R220K variant. The S70 nucleophile attacks the β-lactam ring. The protonation water molecule is closely anchored by E276 and clavulanic acid, thus promoting secondary ring opening and protonation of the oxonium ion intermediate to form the imine. The imine is mostly converted to the inactivation products: an enoyl ether which decomposes to the propynyl enzyme and an aldehyde which gets hydrated to generate a hydrated aldehyde. A minor fraction may tautomerize to the enamine form; thus, it is not represented.