Biomimetic hydrolysis of penicillin G catalyzed by dinuclear zinc(II) complexes: structure-activity correlations in beta-lactamase model systems

A series of highly preorganized pyrazolate-based dinuclear zinc complexes has been studied as functional synthetic analogues of metallo-beta-lactamases, a class of bacterial enzymes that cause serious clinical problems because of their degradation of common beta-lactam antibiotics. We have investigated the hydrolytic cleavage of penicillin G mediated by the different dinuclear zinc complexes, and have deduced structure-activity correlations. While cooperative effects of the adjacent metal ions might be operative, these are found to either enhance or diminish beta-lactamase activity with respect to a single free zinc. Drastic differences in activity are ascribed to a lack of accessible binding sites after incorporation of the substrate within the bimetallic pocket of 2 and 4, whereas partial detachment of hemilabile ligand side arms in 1 and 3 opens up available coordination sites for nucleophile activation and/or for binding and polarisation of the beta-lactam amide oxygen atom. This interpretation has been corroborated by NMR spectroscopic and mass spectrometric evidence as well as by X-ray crystallography of several adducts formed between the pyrazolate-based dinuclear zinc scaffolds and the small substrate analogue oxazetidinylacetate (oaa), 5-7. In all adducts, the carboxylate group of oaa is the primary anchoring site and is nested in a bridging position within the bimetallic pocket. However, zinc binding of the beta-lactam amide oxygen atom has been confirmed crystallographically for the first time in 7, in which additional open-site coordination sites are available.     

Conformational changes in the metallo-beta-lactamase ImiS during the catalytic reaction: an EPR spectrokinetic study of Co(II)-spin label interactions

Metallo-beta-lactamases are responsible for conferring antibiotic resistance on certain pathogenic bacteria. In consequence, the search for inhibitors that may be useful in combating antibiotic resistance has fueled much study of the active sites of these enzymes. There exists circumstantial evidence that the binding of substrates and inhibitors to metallo-beta-lactamases may involve binding to the organic part of the molecule, in addition to or prior to binding to one or more active site metal ions. It has also been postulated that a conformational change may accompany this putative binding. In the present study, electron paramagnetic resonance spectrokinetic study of a spin-labeled variant of the class B2 metallo-beta-lactamase ImiS identified movement of a component residue on a conserved alpha-helix in a catalytically competent time upon formation of a transient reaction intermediate with the substrate imipenem. In a significant subpopulation of ImiS, this conformational change was not associated with substrate binding to the active site metal ion but, rather, represents a distinct step in the reaction with ImiS. This observation has implications regarding the determinants of substrate specificity in metallo-beta-lactamases and the design of potentially clinically useful inhibitors.     

Metal content and localization during turnover in B. cereus metallo-beta-lactamase

Metallo-beta-lactamases are enzymes capable of hydrolyzing all known classes of beta-lactam antibiotics, rendering them ineffective. The design of inhibitors active against all classes of metallo-beta-lactamases has been hampered by the heterogeneity in metal content in the active site and the existence of two different mononuclear forms. BcII is a B1 metallo-beta-lactamase which is found in both mononuclear and dinuclear forms. Despite very elegant studies, there is still controversy on the nature of the active BcII species. We carried out a non-steady-state study of the hydrolysis of penicillin G catalyzed by Co(II)-substituted BcII, and we followed the modifications occurring at the active site of the enzyme. Working at different metal/enzyme ratios we demonstrate that both mono-Co(II) and di-Co(II) BcII are active metallo-beta-lactamases. Besides, we here present evidence that during penicillin G hydrolysis catalyzed by mono-Co(II) BcII the metal is localized in the DCH site (the Zn2 site in B1 enzymes). These conclusions allow us to propose that both in mono-Co(II) and di-Co(II) BcII the substrate is bound to the enzyme through interactions with the Co(II) ion localized in the DCH site. The finding that the DCH site is able to give rise to an active lactamase suggests that the Zn2 site is a common feature to all subclasses of metallo-beta-lactamases and would play a similar role. This proposal provides a starting point for the design of inhibitors based on transition-state analogs, which might be effective against all MbetaLs.     

Trapping and characterization of a reaction intermediate in carbapenem hydrolysis by B. cereus metallo-beta-lactamase

Metallo-beta-lactamases hydrolyze most beta-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)-BcII with imipenem. These studies show that Co(II)-BcII is able to hydrolyze imipenem in both the mono- and dinuclear forms. In contrast to the situation met for penicillin, the species that accumulates during turnover is an enzyme-intermediate adduct in which the beta-lactam bond has already been cleaved. This intermediate is a metal-bound anionic species with a novel resonant structure that is stabilized by the metal ion at the DCH or Zn2 site. This species has been characterized based on its spectroscopic features. This represents a novel, previously unforeseen intermediate that is related to the chemical nature of carbapenems, as confirmed by the finding of a similar intermediate for meropenem. Since carbapenems are the only substrates cleaved by B1, B2, and B3 lactamases, identification of this intermediate could be exploited as a first step toward the design of transition-state-based inhibitors for all three classes of metallo-beta-lactamases.     

Energetic, structural, and antimicrobial analyses of beta-lactam side chain recognition by beta-lactamases

BACKGROUND: Penicillins and cephalosporins are among the most widely used and successful antibiotics. The emergence of resistance to these beta-lactams, most often through bacterial expression of beta-lactamases, threatens public health. To understand how beta-lactamases recognize their substrates, it would be helpful to know their binding energies. Unfortunately, these have been difficult to measure because beta-lactams form covalent adducts with beta-lactamases. This has complicated functional analyses and inhibitor design. RESULTS: To investigate the contribution to interaction energy of the key amide (R1) side chain of beta-lactam antibiotics, eight acylglycineboronic acids that bear the side chains of characteristic penicillins and cephalosporins, as well as four other analogs, were synthesized. These transition-state analogs form reversible adducts with serine beta-lactamases. Therefore, binding energies can be calculated directly from K(i) values. The K(i) values measured span four orders of magnitude against the Group I beta-lactamase AmpC and three orders of magnitude against the Group II beta-lactamase TEM-1. The acylglycineboronic acids have K(i) values as low as 20 nM against AmpC and as low as 390 nM against TEM-1. The inhibitors showed little activity against serine proteases, such as chymotrypsin. R1 side chains characteristic of beta-lactam inhibitors did not have better affinity for AmpC than did side chains characteristic of beta-lactam substrates. Two of the inhibitors reversed the resistance of pathogenic bacteria to beta-lactams in cell culture. Structures of two inhibitors in their complexes with AmpC were determined by X-ray crystallography to 1.90 A and 1.75 A resolution; these structures suggest interactions that are important to the affinity of the inhibitors. CONCLUSIONS: Acylglycineboronic acids allow us to begin to dissect interaction energies between beta-lactam side chains and beta-lactamases. Surprisingly, there is little correlation between the affinity contributed by R1 side chains and their occurrence in beta-lactam inhibitors or beta-lactam substrates of serine beta-lactamases. Nevertheless, presented in acylglycineboronic acids, these side chains can lead to inhibitors with high affinities and specificities. The structures of their complexes with AmpC give a molecular context to their affinities and may guide the design of anti-resistance compounds in this series.     

Monitoring the zinc affinity of the metallo-β-lactamase CphA by automated nanoESI-MS

Metallo-beta-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate beta-lactam antibiotics. The subclass B2 enzyme CphA of Aeromonas hydrophila is a unique metallo-beta-lactamase because it degrades only carbapenems efficiently and is only active when it has one zinc ion bound. A zinc titration experiment was used to study the zinc affinity of the wild-type and of several mutant CphA enzymes. It shows that a second Zn(2+) is also bound at high ion concentrations. All samples were analyzed using mass spectrometry in combination with an automated nanoESI source. The metal-free enzyme has a bimodal charge distribution indicative of two conformational states. A completely folded enzyme is detected when the apo-enzyme has bound the first zinc. Intensity ratios of the different enzyme forms were used to deduce the zinc affinities. CphA enzymes mutated in metal ligands show decreased zinc affinity compared to wild-type, especially D120 mutants.     

Inhibitor binding by metallo-beta-lactamase IMP-1 from Pseudomonas aeruginosa: quantum mechanical/molecular mechanical simulations

The dynamics of the IMP-1 enzyme complexed with three prototypical inhibitors are investigated using a quantum mechanical/molecular mechanical (QM/MM) method based on the self-consistent-charge density-functional tight-binding model. The binding patterns of the inhibitors observed in X-ray diffraction experiments are well reproduced in 600 ps molecular dynamics simulations at room temperature. These inhibitors anchor themselves in the enzyme active site by direct coordination with the two zinc ions, displacing the hydroxide nucleophile that bridges the two zinc ions. In addition, they also interact with several active-site residues and those in two mobile loops. The excellent agreement with experimental structural data validates the QM/MM treatment used in our simulations.     

Catalysis of hydrolysis and aminolysis of non-classical beta-lactam antibiotics by metal ions and metal chelates.

The Zn(2+)-tris (hydroxymethyl)aminomethane (Tris) system has a great catalytic effect on the hydrolysis and aminolysis of some beta-lactam antibiotics. In order to ascertain the mechanism of this catalysis we have analysed the effects of the beta-lactam antibiotic structure. First we studied the kinetics of the decomposition of imipenem, SCH 29482, aztreonam and nocardicin A in aqueous solution of Tris at 35.0 degrees C, 0.5 mol.dm-3 ionic strength and in the presence of metal ions (Zn2+, Cd2+, Co2+, Cu2+, Ni2+ and Mn2+). From these studies, we conclude that Tris and metal ions (in separate solutions) exert a great catalytic effect on the hydrolysis of imipenem and SCH 29482. We suggest that in metal ion solutions a 1:1 complex is formed between the metal ion and beta-lactam antibiotic, which is attacked by hydroxide ions. Studies of the degradation of the antibiotics studied in solutions of Tris and metal ions together indicate that the systems Cd(2+)-Tris and Zn(2+)-Tris have a great catalytic effect on the hydrolysis and aminolysis of imipenem and SCH 29482. We suggest that this catalysis takes place via a ternary complex in which the metal ion plays a double role by (a) placing the antibiotic and the Tris in the right position for the reaction and (b) lowering the pKa of the hydroxide group of Tris, which is coordinated with the metal ion, generating a strong nucleophile.     

Rational design of a novel fluorescent biosensor for beta-lactam antibiotics from a class A beta-lactamase

A rational design strategy was used to construct a sensitive "turn-on" biosensor for beta-lactam antibiotics and beta-lactamase inhibitors from a class A beta-lactamase mutant with suppressed hydrolytic activity. A fluorescein molecule was attached to the 166 position on the Omega-loop of the E166C mutant close to the active site of the beta-lactamase. Upon binding with antibiotics or inhibitors, the flexibility of the Omega-loop allows the fluorescein molecule to move out from the active site and be more exposed to solvent. This process is accompanied by an increase in the fluorescence of the labeled enzyme. The fluorescence intensity of the biosensor increases with the concentration of antibiotics or inhibitors, which can detect penicillin G at concentrations as low as 50 nM in water. This approach opens a possibility for converting highly active and nonallosteric enzymes into substrate-binding proteins for biosensing purposes.     

Studies on ternary metallo-β lactamase-inhibitor complexes using electrospray ionization mass spectrometry

Metallo-beta-lactamases (MBLs) are targets for medicinal chemistry as they mediate bacterial resistance to beta-lactam antibiotics. Electrospray-ionization mass spectrometry (ESI-MS) was used to study the inhibition by a set of mercaptocarboxylates of two representative MBLs with different optimal metal stoichiometries for catalysis. BcII is a dizinc MBL (Class B1), whilst the CphA MBL (Class B2) exhibits highest activity with a single zinc ion in the active site. Experimental parameters for the detection of the metallo-enzyme and the metallo-enzyme-inhibitor complexes were evaluated and optimized. Following investigations on the stoichiometry of metal binding, the affinity of the inhibitors was investigated by measuring the relative abundance of the complex compared to the metalloprotein. The results for the BcII enzyme were in general agreement with solution assays and demonstrated that the inhibitors bind to the dizinc form of the BcII enzyme. The results for the CphA(ZnII) complex unexpectedly revealed an increased affinity for the binding of a second metal ion in the presence of thiomandelic acid. The results demonstrate that direct ESI-MS analysis of enzyme:inhibitor complexes is a viable method for screening inhibitors and for the rapid assay of the enzyme:metal:inhibitor ratios.     

Role of zinc content on the catalytic efficiency of B1 metallo beta-lactamases

Metallo beta-lactamases (MbetaL) are enzymes naturally evolved by bacterial strains under the evolutionary pressure of beta-lactam antibiotic clinical use. They have a broad substrate spectrum and are resistant to all the clinically useful inhibitors, representing a potential risk of infection if massively disseminated. The MbetaL scaffold is designed to accommodate one or two zinc ions able to activate a nucleophilic hydroxide for the hydrolysis of the beta-lactam ring. The role of zinc content on the binding and reactive mechanism of action has been the subject of debate and still remains an open issue despite the large amount of data acquired. We report herein a study of the reaction pathway for binuclear CcrA from Bacteroides fragilis using density functional theory based quantum mechanics-molecular mechanics dynamical modeling. CcrA is the prototypical binuclear enzyme belonging to the B1 MbetaL family, which includes several harmful chromosomally encoded and transferable enzymes. The involvement of a second zinc ion in the catalytic mechanism lowers the energetic barrier for beta-lactam hydrolysis, preserving the essential binding features found in mononuclear B1 enzymes (BcII from Bacillus cereus) while providing a more efficient single-step mechanism. Overall, this study suggests that uptake of a second equivalent zinc ion is evolutionary favored.     

Activation for catalysis of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by bacterial cell wall

Methicillin-resistant Staphylococcus aureus (MRSA) has acquired a unique penicillin-binding protein (PBP), PBP 2a, which has rendered the organism resistant to the action of all available beta-lactam antibiotics. The X-ray structure of PBP 2a shows the active site in a closed conformation, consistent with resistance to inhibition by beta-lactam antibiotics. However, it is known that PBP 2a avidly cross-links the S. aureus cell wall, which is its physiological function. It is shown herein that synthetic fragments of the bacterial cell wall bind in a saturable manner to PBP 2a and cause a conformational change in the protein that makes the active site more accessible to binding to a beta-lactam antibiotic. These observations and measurements point to a novel strategy by nature to keep the active site of PBP 2a sheltered from the inhibitory activity of the antibiotics, yet it becomes available to the polymeric cell wall by a requisite conformational change for the critical cell wall cross-linking reaction.     

Water-assisted reaction mechanism of monozinc beta-lactamases

Hybrid Car-Parrinello QM/MM calculations are used to investigate the reaction mechanism of hydrolysis of a common beta-lactam substrate (cefotaxime) by the monozinc beta-lactamase from Bacillus cereus (BcII). The calculations suggest a fundamental role for an active site water in the catalytic mechanism. This water molecule binds the zinc ion in the first step of the reaction, expanding the zinc coordination number and providing a proton donor adequately oriented for the second step. The free energy barriers of the two reaction steps are similar and consistent with the available experimental data. The conserved hydrogen bond network in the active site, defined by Asp120, Cys221, and His263, not only contributes to orient the nucleophile (as already proposed), but it also guides the second catalytic water molecule to the zinc ion after the substrate is bound. The hydrolysis reaction in water has a relatively high free energy barrier, which is consistent with the stability of cefotaxime in water solution. The modeled Michaelis complexes for other substrates are also characterized by the presence of an ordered water molecule in the same position, suggesting that this mechanism might be general for the hydrolysis of different beta-lactam substrates.     

Role of the Zn1 and Zn2 sites in metallo-beta-lactamase L1

In an effort to probe the role of the Zn(II) sites in metallo-beta-lactamase L1, mononuclear metal ion containing and heterobimetallic analogues of the enzyme were generated and characterized using kinetic and spectroscopic studies. Mononuclear Zn(II)-containing L1, which binds Zn(II) in the consensus Zn1 site, was shown to be slightly active; however, this enzyme did not stabilize a nitrocefin-derived reaction intermediate that had been previously detected. Mononuclear Co(II)- and Fe(III)-containing L1 were essentially inactive, and NMR and EPR studies suggest that these metal ions bind to the consensus Zn2 site in L1. Heterobimetallic analogues (ZnCo and ZnFe) analogues of L1 were generated, and stopped-flow kinetic studies revealed that these enzymes rapidly hydrolyze nitrocefin and that there are large amounts of the reaction intermediate formed during the reaction. The heterobimetallic analogues were reacted with nitrocefin, and the reactions were rapidly freeze quenched. EPR studies on these samples demonstrate that Co(II) is 5-coordinate in the resting state, proceeds through a 4-coordinate species during the reaction, and is 5-coordinate in the enzyme-product complex. These studies demonstrate that the metal ion in the Zn1 site is essential for catalysis in L1 and that the metal ion in the Zn2 site is crucial for stabilization of the nitrocefin-derived reaction intermediate.     

Two-Metal Ion Catalysis in Enzymatic Acyl- and Phosphoryl-Transfer Reactions

Numerous studies, both in enzymatic and nonenzymatic catalysis, have been undertaken to understand the way by which metal ions, especially zinc ions, promote the hydrolysis of phosphate ester and amide bonds. Hydrolases containing one metal ion in the active site, termed mononuclear metallohydrolases, such as carboxypeptidase. A and thermolysin were among the first enzymes to have their structures unraveled by X-ray crystallography. In recent years an increasing number of metalloenzymes have been identified that use two or more adjacent metal ions in the catalysis of phosphoryl-transfer reactions (R-OPO₃ + R′-OH → R′-OPO₃ + R-OH; in the case of the phosphatase reaction R′-OH is a water molecule) and carbonyl-transfer reactions, for example, in peptidases or other amidases. These dinuclear metalloenzymes catalyze a great variety of these reactions, including hydrolytic cleavage of phosphomono-, -di- and -triester bonds, phosphoanhydride bonds as well as of peptide bonds or urea. In addition, the formation of the phosphodiester bond of RNA and DNA by polymerases is catalyzed by a two-metal ion mechanism. A remarkable diversity is also seen in the structures of the active sites of these di- and trinuclear metalloenzymes, even for enzymes that catalyze very similar reactions. The determination of the structure of a substrate, product, stable intermediate, or a reaction coordinate analogue compound bound to an active or inactivated enzyme is a powerful approach to investigate mechanistic details of enzyme action. Such studies have been applied to several of the metalloenzymes reviewed in this article; together with many other biochemical studies they provide a growing body of information on how the two (or more) metal ions cooperate to achieve efficient catalysis.     

Zinc-bound thiolate-disulfide exchange: a strategy for inhibiting metallo-beta-lactamases

The mononuclear zinc thiolate complexes [(Tp(PhMe))Zn(S-R)], where Tp(PhMe) is hydrotris((3-methyl-5-phenyl)pyrazolyl)borate and (S-R) is benzyl thiolate, 4-nitrophenylthiolate, 4-trifluoromethylphenylthiolate, 4-chlorophenylthiolate, phenylthiolate, 2-methylphenylthiolate, 4-methylphenylthiolate, 4-methoxyphenylthiolate, or 4-hydroxyphenylthiolate, were synthesized. Representative members of the class were also characterized structurally. The benzyl thiolate complex undergoes a thiolate-disulfide exchange reaction with a variety of diphenyl and dipyridyl disulfides. Kinetic studies revealed that the reaction shows saturation behavior in both complex and disulfide for most of the disulfides studied. Combined with studies of the lability of the coordinated thiolate, a mechanism is proposed where the reactive species is the zinc-coordinated thiolate. When the free benzyl thiol was allowed to react with the same disulfides, the reaction was slower by a factor of 20-200 than that for the zinc-thiolate complex, depending on the particular disulfide employed. Since most metallo-beta-lactamases contain one or more cysteine residues, the one in the active site being coordinated to zinc, the present study was extended to examine whether disulfides can be used as inhibitors of these enzymes by selective oxidation of the metal-bound cysteine. Several disulfides allowed to react with metallo-beta-lactamase CcrA from Bacteroides fragilis were moderate to potent irreversible inhibitors of the enzyme.     

Antibiotic deactivation by a dizinc beta-lactamase: mechanistic insights from QM/MM and DFT studies

Hybrid quantum mechanical/molecular mechanical (QM/MM) methods and density functional theory (DFT) were used to investigate the initial ring-opening step in the hydrolysis of moxalactam catalyzed by the dizinc L1 beta-lactamase from Stenotrophomonas maltophilia. Anchored at the enzyme active site via direct metal binding as suggested by a recent X-ray structure of an enzyme-product complex (Spencer, J.; et al. J. Am. Chem. Soc. 2005, 127, 14439), the substrate is well aligned with the nucleophilic hydroxide that bridges the two zinc ions. Both QM/MM and DFT results indicate that the addition of the hydroxide nucleophile to the carbonyl carbon in the substrate lactam ring leads to a metastable intermediate via a dominant nucleophilic addition barrier. The potential of mean force obtained by SCC-DFTB/MM simulations and corrected by DFT/MM calculations yields a reaction free energy barrier of 23.5 kcal/mol, in reasonable agreement with the experimental value of 18.5 kcal/mol derived from kcat of 0.15 s(-1). It is further shown that zinc-bound Asp120 plays an important role in aligning the nucleophile, but accepts the hydroxide proton only after the nucleophilic addition. The two zinc ions are found to participate intimately in the catalysis, consistent with the proposed mechanism. In particular, the Zn(1) ion is likely to serve as an "oxyanion hole" in stabilizing the carbonyl oxygen, while the Zn(2) ion acts as an electrophilic catalyst to stabilize the anionic nitrogen leaving group.     

The mononuclear metal center of type-I dihydroorotase from aquifex aeolicus

Background

Dihydroorotase (DHO) is a zinc metalloenzyme, although the number of active site zinc ions has been controversial. E. coli DHO was initially thought to have a mononuclear metal center, but the subsequent X-ray structure clearly showed two zinc ions, α and β, at the catalytic site. Aquifex aeolicus DHO, is a dodecamer comprised of six DHO and six aspartate transcarbamoylase (ATC) subunits. The isolated DHO monomer, which lacks catalytic activity, has an intact α-site and conserved β-site ligands, but the geometry of the second metal binding site is completely disrupted. However, the putative β-site is restored when the complex with ATC is formed and DHO activity is regained. Nevertheless, the X-ray structure of the complex revealed a single zinc ion at the active site. The structure of DHO from the pathogenic organism, S. aureus showed that it also has a single active site metal ion.

Results

Zinc analysis showed that the enzyme has one zinc/DHO subunit and the addition of excess metal ion did not stimulate catalytic activity, nor alter the kinetic parameters. The metal free apoenzyme was inactive, but the full activity was restored upon the addition of one equivalent of Zn²⁺ or Co²⁺. Moreover, deletion of the β-site by replacing the His180 and His232 with alanine had no effect on catalysis in the presence or absence of excess zinc. The 2.2 Å structure of the double mutant confirmed that the β-site was eliminated but that the active site remained otherwise intact.

Conclusions

Thus, kinetically competent A. aeolicus DHO has a mononuclear metal center. In contrast, elimination of the putative second metal binding site in amidohydrolyases with a binuclear metal center, resulted in the abolition of catalytic activity. The number of active site metal ions may be a consideration in the design of inhibitors that selectively target either the mononuclear or binuclear enzymes.

     

Novel Chromogenic Substrate for Bacterial β-Lactamases Based on Cephalosporin Modified with an Epoxy Group

Beta-lactamases are the key enzymes involved in resistance to beta-lactam antibiotics in pathogenic bacteria causing infectious diseases. The search for new inhibitors and the study of the resistance mechanisms require the production of chromogenic substrates for beta-lactamases. A novel cephalosporin derivative with an epoxy functional group named CMPD1 is synthesized. It is shown to be a substrate for TEM type beta-lactamases, which is hydrolyzed to form a colored product. The hydrolysis product has an optical absorption maximum at 450 nm. The difference in the absorption maxima of the substrate and the product is 95 nm, and, therefore, CMPD1 exceeds the previously described substrates, according to this parameter. It has been found that the CMPD1 compound is hydrolyzed only by the TEM type beta-lactamases that lack mutations in the active site. This can be used to study the mechanisms of the catalytic effect of beta-lactamases.     

Probing substrate binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis

Background  

The metallo-β-lactamases are Zn(II)-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics.