KPC-2 β-lactamase enables carbapenem antibiotic resistance through fast deacylation of the covalent intermediate
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.015050
ISSN1083-351X
AutoresShrenik Mehta, I. Furey, O.A. Pemberton, David M. Boragine, Yu Chen, Timothy Palzkill,
Tópico(s)Enzyme Structure and Function
ResumoSerine active-site β-lactamases hydrolyze β-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 β-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase. Serine active-site β-lactamases hydrolyze β-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 β-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase. The carbapenem class of β-lactam antibiotics are effective in treating severe infections caused by Gram-negative bacterial pathogens such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (1Papp-Wallace K.M. Endimiani A. Taracila M.A. Bonomo R.A. Carbapenems: past, present, and future.Antimicrob. Agents Chemother. 2011; 55: 4943-4960Crossref PubMed Scopus (655) Google Scholar). Formerly, the carbapenems were reserved as the last line of therapy and were used upon failure of other treatment options (1Papp-Wallace K.M. Endimiani A. Taracila M.A. Bonomo R.A. Carbapenems: past, present, and future.Antimicrob. Agents Chemother. 2011; 55: 4943-4960Crossref PubMed Scopus (655) Google Scholar). However, with increased resistance to other β-lactam classes such as penicillins and cephalosporins, the frequency of carbapenem administration for treating clinical infections is increasing (2Wilson A.P.R. Sparing carbapenem usage.J. Antimicrob. Chemother. 2017; 72: 2410-2417Crossref PubMed Scopus (31) Google Scholar). β-Lactam antibiotics, including carbapenems, act by binding and inactivating a group of essential enzymes, the penicillin binding proteins, resulting in inhibition of peptidoglycan synthesis and ultimately cell death (3Frère J.-M. Page M.G.P. Penicillin-binding proteins: evergreen drug targets.Curr. Opin. Pharmacol. 2014; 18: 112-119Crossref PubMed Scopus (28) Google Scholar). Carbapenems are characterized by a 4:5 fused lactam ring system with an unsaturated C2-C3 bond (Fig. 1) (1Papp-Wallace K.M. Endimiani A. Taracila M.A. Bonomo R.A. Carbapenems: past, present, and future.Antimicrob. Agents Chemother. 2011; 55: 4943-4960Crossref PubMed Scopus (655) Google Scholar). However, the most distinguishing feature of this class is the 6α-1R-hydroxyethyl side chain at position C6 (Fig. 1) (4Birnbaum J. Kahan F.M. Kropp H. MacDonald J.S. Carbapenems, a new class of beta-lactam antibiotics. Discovery and development of imipenem/cilastatin.Am. J. Med. 1985; 78: 3-21Abstract Full Text PDF PubMed Scopus (235) Google Scholar). Conventional β-lactam antibiotics such as penicillins and cephalosporins have a 6β-acylamide substituent at this position (Fig. 1). This change in stereochemistry at the C6 position is important for the potency and broad-spectrum activity of the carbapenem antibiotics (5Maveyraud L. Mourey L. Kotra L.P. Pedelacq J.-D. Guillet V. Mobashery S. Samama J.-P. Structural basis for clinical longevity of carbapenem antibiotics in the face of challenge by the common class A β-lactamases from the antibiotic-resistant bacteria.J. Am. Chem. Soc. 1998; 120: 9748-9752Crossref Scopus (119) Google Scholar, 6Nukaga M. Bethel C.R. Thomson J.M. Hujer A.M. Distler A. Anderson V.E. Knox J.R. Bonomo R.A. Inhibition of class A β-lactamases by carbapenems: crystallographic observation of two conformations of meropenem in SHV-1.J. Am. Chem. Soc. 2008; 130: 12656-12662Crossref PubMed Scopus (52) Google Scholar, 7Fonseca F. Chudyk E.I. van der Kamp M.W. Correia A. Mulholland A.J. Spencer J. The basis for carbapenem hydrolysis by class A β-lactamases: a combined investigation using crystallography and simulations.J. Am. Chem. Soc. 2012; 134: 18275-18285Crossref PubMed Scopus (55) Google Scholar). One of the most important characteristics of carbapenems is that they are poorly hydrolyzed by most β-lactamases (1Papp-Wallace K.M. Endimiani A. Taracila M.A. Bonomo R.A. Carbapenems: past, present, and future.Antimicrob. Agents Chemother. 2011; 55: 4943-4960Crossref PubMed Scopus (655) Google Scholar). The most common mechanism of resistance to β-lactam antibiotics in Gram-negative bacteria is the production of β-lactamases. There are four classes of β-lactamases (A, B, C, D) based on the primary amino acid sequence homology (8Ambler R.P. The structure of beta-lactamases.Phil. Trans. R. Soc. Lond. B Biol. Sci. 1980; 289: 321-331Crossref PubMed Scopus (1259) Google Scholar). Classes A, C, and D are serine hydrolases, whereas class B consists of zinc metalloenzymes. Class B enzymes, including the New Delhi metallo-β-lactamase, can efficiently hydrolyze carbapenems, whereas most of the serine hydrolases do not (9Palzkill T. Metallo-beta-lactamase structure and function.Ann. N. Y. Acad. Sci. 2013; 1277: 91-104Crossref PubMed Scopus (271) Google Scholar). In fact, carbapenems inhibit many clinically important class A β-lactamase enzymes such as TEM-1, SHV-1, and Mycobacterium tuberculosis BlaC through formation of a stable, long-lived, acyl-enzyme complex (5Maveyraud L. Mourey L. Kotra L.P. Pedelacq J.-D. Guillet V. Mobashery S. Samama J.-P. Structural basis for clinical longevity of carbapenem antibiotics in the face of challenge by the common class A β-lactamases from the antibiotic-resistant bacteria.J. Am. Chem. Soc. 1998; 120: 9748-9752Crossref Scopus (119) Google Scholar, 6Nukaga M. Bethel C.R. Thomson J.M. Hujer A.M. Distler A. Anderson V.E. Knox J.R. Bonomo R.A. Inhibition of class A β-lactamases by carbapenems: crystallographic observation of two conformations of meropenem in SHV-1.J. Am. Chem. Soc. 2008; 130: 12656-12662Crossref PubMed Scopus (52) Google Scholar, 10Tremblay L.W. Fan F. Blanchard J.S. Biochemical and structural characterization of Mycobacterium tuberculosis β-lactamase with the carbapenems ertapenem and doripenem.Biochemistry. 2010; 49: 3766-3773Crossref PubMed Scopus (63) Google Scholar, 11Easton C.J. Knowles J.R. Inhibition of the RTEM beta-lactamase from Escherichia coli. Interaction of the enzyme with derivatives of olivanic acid.Biochemistry. 1982; 21: 2857-2862Crossref PubMed Scopus (55) Google Scholar). Several serine β-lactamases belonging to classes A and D, however, have emerged that can hydrolyze carbapenems. Among the class A β-lactamases, several enzymes including KPC, SFC, NMC, IMI, SME that possess carbapenemase activity have been identified in environmental and pathogenic organisms (12Naas T. Vandel L. Sougakoff W. Livermore D.M. Nordmann P. Cloning and sequence analysis of the gene for a carbapenem-hydrolyzing class A beta-lactamase, Sme-1, from Serratia marcescens S6.Antimicrob. Agents Chemother. 1994; 38: 1262-1270Crossref PubMed Scopus (169) Google Scholar, 13Majiduddin F.K. Palzkill T. Amino acid residues that contribute to substrate specificity of class A beta-lactamase SME-1.Antimicrob. Agents Chemother. 2005; 49: 3421-3427Crossref PubMed Scopus (35) Google Scholar, 14Mariotte-Boyer S. Nicolas-Chanoine M.H. Labia R. A kinetic study of NMC-A beta-lactamase, an ambler class A carbapenemase also hydrolyzing cephamycins.FEMS Microbiol. Lett. 1996; 143: 29-33PubMed Google Scholar, 15Mourey L. Miyashita K. Swaren P. Bulychev A. Samama J.P. Mobashery S. Inhibition of the NMC-A beta-lactamase by a penicillanic acid derivative and the structural bases for the increase in substrate profile of this antibiotic resistance enzyme.J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (49) Google Scholar, 16Yigit H. Queenan A.M. Anderson G.J. Domenech-Sanchez A. Biddle J.W. Steward C.D. Alberti S. Bush K. Tenover F.C. Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem- resistant strain of Klebsiella pneumoniae.Antimicrob. Agents Chemother. 2001; 45: 1151-1161Crossref PubMed Scopus (1156) Google Scholar, 17Henriques I. Moura A. Alves A. Saavedra M.J. Correia A. Molecular characterization of a carbapenem-hydrolyzing class A beta-lactamase, SFC-1, from Serratia fonticola UTAD54.Antimicrob. Agents Chemother. 2004; 48: 2321-2324Crossref PubMed Scopus (57) Google Scholar, 18Fonseca F. Sarmento A.C. Henriques I. Samyn B. van Beeumen J. Domingues P. Domingues M.R. Saavedra M.J. Correia A. Biochemical characterization of SFC-1, a class A carbapenem-hydrolyzing beta-lactamase.Antimicrob. Agents Chemother. 2007; 51: 4512-4514Crossref PubMed Scopus (22) Google Scholar). In particular, KPC-producing organisms are now widespread worldwide (19Hidalgo-Grass C. Warburg G. Temper V. Benenson S. Moses A.E. Block C. Strahilevitz J. KPC-9, a novel carbapenemase from clinical specimens in Israel.Antimicrob. Agents Chemother. 2012; 56: 6057-6059Crossref PubMed Scopus (21) Google Scholar, 20Lamoureaux T.L. Frase H. Antunes N.T. Vakulenko S.B. Antibiotic resistance and substrate profiles of the class A carbapenemase KPC-6.Antimicrob. Agents Chemother. 2012; 56: 6006-6008Crossref PubMed Scopus (9) Google Scholar, 21Lascols C. Hackel M. Hujer A.M. Marshall S.H. Bouchillon S.K. Hoban D.J. Hawser S.P. Badal R.E. Bonomo R.A. Using nucleic acid microarrays to perform molecular epidemiology and detect novel β-lactamases: a snapshot of extended-spectrum β-lactamases throughout the world.J. Clin. Microbiol. 2012; 50: 1632-1639Crossref PubMed Scopus (52) Google Scholar, 22Robledo I.E. Aquino E.E. Santé M.I. Santana J.L. Otero D.M. León C.F. Vázquez G.J. Detection of KPC in Acinetobacter spp. in Puerto Rico.Antimicrob. Agents Chemother. 2010; 54: 1354-1357Crossref PubMed Scopus (167) Google Scholar, 23Woodford N. Tierno P.M. Young K. Tysall L. Palepou M.-F.I. Ward E. Painter R.E. Suber D.F. Shungu D. Silver L.L. Inglima K. Kornblum J. Livermore D.M. Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A beta-lactamase, KPC-3, in a New York Medical Center.Antimicrob. Agents Chemother. 2004; 48: 4793-4799Crossref PubMed Scopus (382) Google Scholar). Class A β-lactamases, including KPC-2, hydrolyze the amide bond in β-lactam antibiotics through sequential acylation and deacylation steps (24Fisher J.F. Mobashery S. Three decades of the class A beta-lactamase acyl-enzyme.Curr. Prot. Pept. Sci. 2009; 10: 401-407Crossref PubMed Scopus (53) Google Scholar) (Fig. 2). The catalytic Ser70 residue attacks the carbonyl carbon of the β-lactam resulting in cleavage of the amide bond and formation of a covalent acyl-enzyme intermediate. Subsequently, Glu166 activates a catalytic water molecule for attack on the carbonyl carbon of the acyl-enzyme leading to hydrolysis of the ester bond between Ser70 and the drug. Deacylation regenerates the active enzyme and releases the inactivated drug (24Fisher J.F. Mobashery S. Three decades of the class A beta-lactamase acyl-enzyme.Curr. Prot. Pept. Sci. 2009; 10: 401-407Crossref PubMed Scopus (53) Google Scholar). These reactions can be represented in a minimal kinetic scheme as shown in Figure 2. Structural and biochemical studies on the reaction of imipenem and meropenem with the TEM-1 and SHV-1 enzymes have provided insights into the mechanism of β-lactamase inhibition by carbapenems (5Maveyraud L. Mourey L. Kotra L.P. Pedelacq J.-D. Guillet V. Mobashery S. Samama J.-P. Structural basis for clinical longevity of carbapenem antibiotics in the face of challenge by the common class A β-lactamases from the antibiotic-resistant bacteria.J. Am. Chem. Soc. 1998; 120: 9748-9752Crossref Scopus (119) Google Scholar, 6Nukaga M. Bethel C.R. Thomson J.M. Hujer A.M. Distler A. Anderson V.E. Knox J.R. Bonomo R.A. Inhibition of class A β-lactamases by carbapenems: crystallographic observation of two conformations of meropenem in SHV-1.J. Am. Chem. Soc. 2008; 130: 12656-12662Crossref PubMed Scopus (52) Google Scholar). Nucleophilic attack by the catalytic serine, Ser70, on the β-lactam results in a carbapenem acyl-enzyme complex that is stable owing to a very slow deacylation reaction (Figs. 2 and 3). A number of studies, including X-ray and Raman crystallography and computational simulations have implicated the following factors in stabilization of the acyl-enzyme intermediate: (1) decreased nucleophilicity of the water molecule responsible for deacylation because of a hydrogen bond formed with the 6α-1R-hydroxyethyl group of the carbapenem (7Fonseca F. Chudyk E.I. van der Kamp M.W. Correia A. Mulholland A.J. Spencer J. The basis for carbapenem hydrolysis by class A β-lactamases: a combined investigation using crystallography and simulations.J. Am. Chem. Soc. 2012; 134: 18275-18285Crossref PubMed Scopus (55) Google Scholar); (2) tautomerization of the acyl-enzyme intermediate from the Δ2 to Δ1 pyrroline form, which is even more slowly deacylated (10Tremblay L.W. Fan F. Blanchard J.S. Biochemical and structural characterization of Mycobacterium tuberculosis β-lactamase with the carbapenems ertapenem and doripenem.Biochemistry. 2010; 49: 3766-3773Crossref PubMed Scopus (63) Google Scholar, 25Kalp M. Carey P.R. Carbapenems and SHV-1 beta-lactamase form different acyl-enzyme populations in crystals and solution.Biochemistry. 2008; 47: 11830-11837Crossref PubMed Scopus (26) Google Scholar) (Fig. 3); (3) disruption of the deacylation transition state due to displacement of the acyl-enzyme carbonyl oxygen from the oxyanion hole (formed by the backbone –NH of Ser70 and Thr237) (5Maveyraud L. Mourey L. Kotra L.P. Pedelacq J.-D. Guillet V. Mobashery S. Samama J.-P. Structural basis for clinical longevity of carbapenem antibiotics in the face of challenge by the common class A β-lactamases from the antibiotic-resistant bacteria.J. Am. Chem. Soc. 1998; 120: 9748-9752Crossref Scopus (119) Google Scholar, 6Nukaga M. Bethel C.R. Thomson J.M. Hujer A.M. Distler A. Anderson V.E. Knox J.R. Bonomo R.A. Inhibition of class A β-lactamases by carbapenems: crystallographic observation of two conformations of meropenem in SHV-1.J. Am. Chem. Soc. 2008; 130: 12656-12662Crossref PubMed Scopus (52) Google Scholar, 26Cortina G.A. Hays J.M. Kasson P.M. Conformational intermediate that controls KPC-2 catalysis and beta-lactam drug resistance.ACS Catal. 2018; 8: 2741-2747Crossref PubMed Scopus (11) Google Scholar). For efficient deacylation, it is essential that all the above-mentioned inhibitory traps be avoided. Failure to avoid these traps, as in the case of TEM-1 and SHV-1, leads to inhibition that is characterized by a very slow deacylation reaction and biphasic, branched kinetic progress curves (27Monks J. Waley S.G. Imipenem as substrate and inhibitor of beta-lactamases.Biochem. J. 1988; 253: 323-328Crossref PubMed Scopus (27) Google Scholar). How then, does KPC-2 avoid the traps discussed above and efficiently deacylate carbapenems? A structural alignment of KPC-2 with the non-carbapenem hydrolyzing TEM-1 β-lactamase reveals an overall broad similarity between the structures with much of the variation due to differences in non–active site loops (Fig. 4A). As seen in Figure 4B, many of the active site residues are conserved between KPC-2 and TEM-1 and occupy similar positions. Although there are not obvious distinctions to explain the carbapenemase activity of KPC-2 versus the lack of activity in TEM-1, subtle alterations in the structure or dynamics of KPC-2 could allow conserved residues to contribute to carbapenem hydrolysis in the context of the KPC-2 enzyme but not in the context of the TEM-1 enzyme (28Majiduddin F.K. Palzkill T. Amino acid sequence requirements at residues 69 and 238 for SME-1 β−lactamase to confer resistance to β-lactam antibiotics.Antimicrob. Agents Chemother. 2003; 47: 1062-1067Crossref PubMed Scopus (36) Google Scholar). For example, the KPC-2 active site is larger and more hydrophobic than non-carbapenemase class A enzymes such as TEM-1, which may result in altered conformations of the hydroxyethyl group that are not accessible to non-carbapenemase enzymes and that do not inactivate the deacylation of water molecule (29Ke W. Bethel C.R. Thomson J.M. Bonomo R.A. van den Akker F. Crystal structure of KPC-2: insights into carbapenemase activity in class A β-lactamases.Biochemistry. 2007; 46: 5732-5740Crossref PubMed Scopus (75) Google Scholar, 30Pemberton O.A. Zhang X. Chen Y. Molecular basis of substrate recognition and product release by the Klebsiella pneumoniae carbapenemase (KPC-2).J. Med. Chem. 2017; 60: 3525-3530Crossref PubMed Scopus (24) Google Scholar). Therefore, we examined the contributions of both conserved and nonconserved active site positions to carbapenem hydrolysis by creating alanine substitutions and characterizing the kinetic properties of the mutated enzymes. Sequence alignments of active-site residues of KPC-2 with other carbapenem-hydrolyzing and non-carbapenem-hydrolyzing β-lactamases reveal that Glu166 and Asn170 are highly conserved among class A β-lactamases (Table 1) where they play a role in deacylation of the acyl-enzyme complex (24Fisher J.F. Mobashery S. Three decades of the class A beta-lactamase acyl-enzyme.Curr. Prot. Pept. Sci. 2009; 10: 401-407Crossref PubMed Scopus (53) Google Scholar, 31Taibi P. Mobashery S. Mechanism of turnover of imipenem by the TEM β-lactamase revisted.J. Am. Chem. Soc. 1995; 117: 7600-7605Crossref Scopus (51) Google Scholar). In addition, analysis of the SFC-1 carbapenemase (69% sequence identity to KPC-2) cocrystal structure in complex with meropenem identified residues Thr235, Thr237, and Arg220, which are not conserved in TEM-1, to play a role in binding of the substrate (7Fonseca F. Chudyk E.I. van der Kamp M.W. Correia A. Mulholland A.J. Spencer J. The basis for carbapenem hydrolysis by class A β-lactamases: a combined investigation using crystallography and simulations.J. Am. Chem. Soc. 2012; 134: 18275-18285Crossref PubMed Scopus (55) Google Scholar) (Fig. 4, D–E). In the SFC-1 S70A meropenem structure (S70A mutation prevents hydrolysis), the side chains of residues Thr235 and Thr237 interact with the C3 carboxyl group while the Thr237 main-chain nitrogen forms the oxyanion hole along with that of residue 70 (7Fonseca F. Chudyk E.I. van der Kamp M.W. Correia A. Mulholland A.J. Spencer J. The basis for carbapenem hydrolysis by class A β-lactamases: a combined investigation using crystallography and simulations.J. Am. Chem. Soc. 2012; 134: 18275-18285Crossref PubMed Scopus (55) Google Scholar). The Arg220 residue forms hydrogen bonds with the Thr237 side chain, and the positive charge associated with the residue may contribute to substrate binding by providing an environment favorable for the negatively charged C3 carboxylate of carbapenems (29Ke W. Bethel C.R. Thomson J.M. Bonomo R.A. van den Akker F. Crystal structure of KPC-2: insights into carbapenemase activity in class A β-lactamases.Biochemistry. 2007; 46: 5732-5740Crossref PubMed Scopus (75) Google Scholar, 30Pemberton O.A. Zhang X. Chen Y. Molecular basis of substrate recognition and product release by the Klebsiella pneumoniae carbapenemase (KPC-2).J. Med. Chem. 2017; 60: 3525-3530Crossref PubMed Scopus (24) Google Scholar). In addition, the structure of KPC-2 in complex with hydrolyzed faropenem identified similar interactions with hydrogen bonds between Thr235 and Thr237 and the C3 carboxylate with Arg220 forming a hydrogen bond with Thr237 (30Pemberton O.A. Zhang X. Chen Y. Molecular basis of substrate recognition and product release by the Klebsiella pneumoniae carbapenemase (KPC-2).J. Med. Chem. 2017; 60: 3525-3530Crossref PubMed Scopus (24) Google Scholar). In order to examine the role of these residues in carbapenem hydrolysis by KPC-2, we created alanine mutants at positions 166, 170, 220, 235, and 237 and used pre-steady-state kinetic analysis to examine their contribution to the acylation and deacylation rates for turnover of the carbapenem, imipenem. In addition, R220Q was created to test the importance of positive charge at this position. The results indicate that, although the deacylation rate is still slower than acylation, wildtype KPC-2 β-lactamase hydrolyzes carbapenems efficiently because of a deacylation rate that is orders of magnitude faster than that previously observed for TEM-1 (31Taibi P. Mobashery S. Mechanism of turnover of imipenem by the TEM β-lactamase revisted.J. Am. Chem. Soc. 1995; 117: 7600-7605Crossref Scopus (51) Google Scholar). In addition, the results show residues Asn170, Arg220, and Thr237 all enhance both the acylation and deacylation rates. Substitution of residue Asn170, however, results in the largest reduction in kcat for imipenem hydrolysis due to a >3000-fold decrease in the deacylation rate. Interestingly, although acylation and deacylation rates are not known, the N170A substitution had only modest effects on kcat for ampicillin and cephalothin catalysis, suggesting that Asn170 is critical for carbapenem but not penicillin or cephalosporin hydrolysis by KPC-2 β-lactamase.Table 1Alignment of select active site residues of common class A β-lactamases and carbapenemasesβ-lactamasePositionkcat (s−1)bkcat values for imipenem hydrolysis. (ref)166170220235237244TEM-1ENLSAR0.003 (31Taibi P. Mobashery S. Mechanism of turnover of imipenem by the TEM β-lactamase revisted.J. Am. Chem. Soc. 1995; 117: 7600-7605Crossref Scopus (51) Google Scholar)SHV-1ENLTARNDSMEaCarbapenemase enzymes.ENRTSA185 (13Majiduddin F.K. Palzkill T. Amino acid residues that contribute to substrate specificity of class A beta-lactamase SME-1.Antimicrob. Agents Chemother. 2005; 49: 3421-3427Crossref PubMed Scopus (35) Google Scholar)NMC-AaCarbapenemase enzymes.ENRTSA130 (15Mourey L. Miyashita K. Swaren P. Bulychev A. Samama J.P. Mobashery S. Inhibition of the NMC-A beta-lactamase by a penicillanic acid derivative and the structural bases for the increase in substrate profile of this antibiotic resistance enzyme.J. Am. Chem. Soc. 1998; 120: 9382-9383Crossref Scopus (49) Google Scholar)SFC-1aCarbapenemase enzymes.ENRTTA54 (18Fonseca F. Sarmento A.C. Henriques I. Samyn B. van Beeumen J. Domingues P. Domingues M.R. Saavedra M.J. Correia A. Biochemical characterization of SFC-1, a class A carbapenem-hydrolyzing beta-lactamase.Antimicrob. Agents Chemother. 2007; 51: 4512-4514Crossref PubMed Scopus (22) Google Scholar)KPC-2aCarbapenemase enzymes.ENRTTA44 (this paper)GES-1EGTTTR0.006 (32Stewart N.K. Smith C.A. Frase H. Black D.J. Vakulenko S.B. Kinetic and structural requirements for carbapenemase activity in GES-type β-lactamases.Biochemistry. 2015; 54: 588-597Crossref PubMed Scopus (24) Google Scholar)GES-2aCarbapenemase enzymes.ENTTTR0.012 (32Stewart N.K. Smith C.A. Frase H. Black D.J. Vakulenko S.B. Kinetic and structural requirements for carbapenemase activity in GES-type β-lactamases.Biochemistry. 2015; 54: 588-597Crossref PubMed Scopus (24) Google Scholar)GES-5aCarbapenemase enzymes.ESTTTR0.44 (32Stewart N.K. Smith C.A. Frase H. Black D.J. Vakulenko S.B. Kinetic and structural requirements for carbapenemase activity in GES-type β-lactamases.Biochemistry. 2015; 54: 588-597Crossref PubMed Scopus (24) Google Scholar)a Carbapenemase enzymes.b kcat values for imipenem hydrolysis. Open table in a new tab In order to examine the basis of carbapenemase activity of KPC-2 and the roles of specific active-site residues in enzyme function, purified wildtype KPC-2 and the E166A, N170A, R220A, R220Q, T235A, and T237A mutant enzymes were subjected to steady-state and pre-steady-state kinetic studies. Traditional steady-state enzyme kinetic parameters were obtained for the hydrolysis of ampicillin, a penicillin, cephalothin, a first-generation cephalosporin, and the carbapenems imipenem and meropenem. Studies with other carbapenemases such as GES-2 and GES-5 have revealed that nonbulky carbapenems such as imipenem have different kinetic parameters compared with bulky carbapenems such as meropenem, doripenem, and ertapenem due to different binding modes in the enzyme active site (32Stewart N.K. Smith C.A. Frase H. Black D.J. Vakulenko S.B. Kinetic and structural requirements for carbapenemase activity in GES-type β-lactamases.Biochemistry. 2015; 54: 588-597Crossref PubMed Scopus (24) Google Scholar) (Fig. 1). Thus, imipenem and meropenem were chosen to represent both nonbulky and bulky carbapenems. Non-carbapenemase class A β-lactamases are inhibited by carbapenems owing to a very slow deacylation rate (5Maveyraud L. Mourey L. Kotra L.P. Pedelacq J.-D. Guillet V. Mobashery S. Samama J.-P. Structural basis for clinical longevity of carbapenem antibiotics in the face of challenge by the common class A β-lactamases from the antibiotic-resistant bacteria.J. Am. Chem. Soc. 1998; 120: 9748-9752Crossref Scopus (119) Google Scholar, 6Nukaga M. Bethel C.R. Thomson J.M. Hujer A.M. Distler A. Anderson V.E. Knox J.R. Bonomo R.A. Inhibition of class A β-lactamases by carbapenems: crystallographic observation of two conformations of meropenem in SHV-1.J. Am. Chem. Soc. 2008; 130: 12656-12662Crossref PubMed Scopus (52) Google Scholar, 27Monks J. Waley S.G. Imipenem as substrate and inhibitor of beta-lactamases.Biochem. J. 1988; 253: 323-328Crossref PubMed Scopus (27) Google Scholar). Therefore, the rates of acylation and deacylation for KPC-2 and the mutants were determined in single turnover pre-steady-state kinetic experiments with imipenem as substrate to examine the rate of acylation and, by inference, the deacylation rate. The wildtype KPC-2 enzyme displayed specificity constants (kcat/KM) of 2.3 × 105, 1.0 × 106, 1.6 × 105, and 1.4 × 105 M−1 s−1 for ampicillin, cephalothin, imipenem, and meropenem hydrolysis, respectively (Table 2) (Figs. S1–S4). Thus, ampicillin, imipenem, and meropenem are hydrolyzed at similar rates, whereas cephalothin is hydrolyzed at an ∼10-fold faster rate. In comparison with the non-carbapenemase TEM-1 enzyme, the specificity constant displayed by KPC-2 for ampicillin hydrolysis is 170-fold lower (33Zafaralla G. Mobashery S. Facilitation of the D2 Æ D1 pyrroline tautomerization of carbapenam antibiotics by the highly conserved arginine-244 of class A β-lactamases during the course of turnover.J. Am. Chem. Soc. 1992; 114: 1505-1506Crossref Scopus (43) Google Scholar). In contrast, the TEM-1 enzyme essentially does not hydrolyze imipenem and meropenem and so KPC-2-mediated hydrolysis of these substrates is much faster.Table 2Steady-state kinetic parameters for KPC-2 and mutant β-lactamasesAMPCEPIMIMEROKPC-2 kcat (s−1)47 ± 3142 ± 1444 ± 13.4 ± 0.1 Km (μM)203 ± 35135 ± 35275 ± 1524 ± 1.1 kcat/Km (μM−1 s−1)0.23 ± 0.041.0 ± 0.20.16 ± 0.010.14 ± 0.04E166A kcat (s−1)HNDaHND, hydrolysis not detected.HNDaHND, hydrolysis not detected.HNDaHND, hydrolysis not detected.HNDaHND, hydrolysis not detected. Km (μM)HNDaHND, hydrolysis not detected.HNDaHND, hydrolysis not detected.HNDaHND, hydrolysis not detected.HNDaHND, hydrolysis not detected. kcat/Km (μM−1 s−1)HNDaHND, hydrolysis not detected.HNDaHND, hydrolysis not detect
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