Meropenem and Chromacef Intermediates Observed in IMP-25 Metallo-β-Lactamase-Catalyzed Hydrolysis

Abstract

Metallo-␤-lactamases (MBLs) efficiently inactivate most ␤-lactam antibacterials and have recently raised concerns due to this broad substrate spectrum, their global spread in various Gram-negative bacteria, and the absence of inhibitors for clinical use (1-3). Zn(II)-bound anionic intermediates of chromogenic ␤-lactams, such as nitrocefin, have been observed during their hydrolysis catalyzed by the MBLs CcrA (4), L1 (5), NDM-1 (6), and VIM-2 (7). For imipenemase (IMP-1), there might be a nitrocefin intermediate (8), although other studies negate this (9, 10). Tioni and coworkers also observed imipenem and meropenem intermediates in BcII (11). Recently, we found that the variant IMP-25 hydrolyzes meropenem more efficiently than IMP-1 and IMP-6 (12). IMP-6 was previously reported to confer resistance to carbapenems, especially meropenem (13). The increasing k cat for meropenem hydrolysis in the order IMP-1 ¡ IMP-6 ¡ IMP-25 (22 Ϯ 1 s Ϫ1 ¡ 60 Ϯ 10 s Ϫ1 ¡ 100 Ϯ 10 s Ϫ1 , respectively) also translates into increasing MICs of meropenem (16 g/ml ¡ 64 g/ml ¡ 128 g/ml, respectively) (12).To study in more detail the basis of the increased k cat in IMP-25 versus IMP-1, we carried out pre-steady-state kinetic experiments (6) for the hydrolysis of meropenem [6°C, 25 M enzyme (E) and substrate (S) ([E]-to-[S] ratio of 1:1) in 50 mM MOPS (morpholinepropanesulfonic acid) (pH 7.0) supplemented with 100 M Zn(II) ions]. The meropenem substrate (S) was tracked at 310 nm (due to noise at 300 nm), product (P) was tracked at 342 nm, and intermediate (I) was tracked at 390 nm. Details on the determination of extinction coefficients and conversion of the spectral data into concentrations are provided in the supplemental material. With IMP-1, a very small amount of intermediate was observed (Fig. 1E, black line), and the data could not be fitted to a two-phase association/decay function. Substrate disappeared at approximately the same exponential rate (25 Ϯ 3 s Ϫ1 ) at which product appeared (38 Ϯ 12 s Ϫ1 ) (Table 1). In contrast, with IMP-25 (Fig. 1A and B and Table 1), an initial appearance of I at 300 Ϯ 100 s Ϫ1 was followed by its disappearance at 24 Ϯ 8 s Ϫ1 . As with IMP-1, the exponential disappearance rate of S (27 Ϯ 1 s Ϫ1 ) was similar to the appearance rate of P (30 Ϯ 20 s Ϫ1 ). Thus, the observed rates of S disappearance and P appearance are indistinguishable between IMP-1 and IMP-25 under these conditions, and the uncertainty of the product formation rates was rather high. However, a clear formation and decay of intermediate was observed in IMP-25. Intermediate accumulated to no more than 6% of the initial [S], which equals [E], meaning that only up to 6% of the E molecules were bound to I.In order to explore if a clearer picture could be obtained at conditions that are more similar to steady-state conditions, we increased [S] to 100 M, 200 M, and 500 M. Several complications became apparent for the accurate quantification of S and P and, consequently, their disappearance and appearance rates, respectively, at higher concentr...

A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases

“…2 ). These results are consistent with the observation of reaction intermediates at 390 and 380 nm in SPM-1 42 and IMP-25 43 , that we are now able to assign to EI 1 . Other enzymes, such as mono-Zn(II) Bla2 did not show accumulation of any intermediate 31 .…”

“…All data here presented strongly support that EI 2 undergoes a stereoselective protonation at C-2 involving a water molecule not bound to the metal site. This mechanistic scheme is fully consistent with all previous experimental evidence 22 , 23 , 28 , 29 , 42 , 43 , 45 . Finally, this mechanism highlights the difference between carbapenem and cephalosphorin hydrolysis.…”

Arginine-containing peptides as potent inhibitors of VIM-2 metallo-β-lactamase

“…However, an absorption band corresponding to a reaction intermediate was not observed (data not shown). This finding is similar to that documented in three studies on IMP-1 [35,37,38], and in investigations on Bla2…”

“…To establish how quickly arginine-containing peptides interact with VIM-2, rapid scanning typically at wavelengths greater than 575 nm [32][33][34][35][36], the stopped-flow experiments were extended to cover the wavelength range from 475 nm to 700 nm. However, an absorption band corresponding to a reaction intermediate was not observed (data not shown).…”

Use of ferrous iron by metallo-β-lactamases

“…In some cases, previous work with nitrocefin, and the related substrate chromacef, has identified an intermediate, absorbing at 665 nm, corresponding to the deprotonated delocalised anion with MBLs including VIM-2, IMP-1, New Delhi metallo-β-lactamase 1 (NDM-1) and L1 [21], [52], [53], [54], [69], [70], [71]. Such a feature was not visible in the di-Zn(II) BcII reaction spectrum, as has been previously reported [56], suggesting that di-Fe(II) BcII may employ a different mechanism for β-lactam hydrolysis (Fig.…”