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Tazobactam Inactivation of SHV-1 and the Inhibitor-resistant Ser130 → Gly SHV-1 β-Lactamase

2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês

10.1074/jbc.m311669200

ISSN

1083-351X

Autores

Doritza Pagán-Rodríguez, Xiang Zhou, Reiko Simmons, Christopher R. Bethel, Andrea M. Hujer, Marion S. Helfand, Zhaoyan Jin, Baochuan Guo, Vernon Anderson, Lily Ng, Robert A. Bonomo,

Tópico(s)

Pneumonia and Respiratory Infections

Resumo

The increasing number of bacteria resistant to combinations of β-lactam and β-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant β-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser130 → Gly inhibitor-resistant variant of SHV-1 β-lactamase were inactivated with tazobactam, a potent class A β-lactamase inhibitor. Apoenzymes and inhibited β-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser130 → Gly (+ Δ 88 Da and + Δ 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive β-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Δ 70 Da) and a hydrated aldehyde (+ Δ 88 Da) as stable products of inhibition. Our results reveal that the Ser → Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser130 → Gly β-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser70 in this enzyme. After acylation of tazobactam by Ser130 → Gly, inactivation proceeds independent of any additional covalent interactions. The increasing number of bacteria resistant to combinations of β-lactam and β-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant β-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser130 → Gly inhibitor-resistant variant of SHV-1 β-lactamase were inactivated with tazobactam, a potent class A β-lactamase inhibitor. Apoenzymes and inhibited β-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser130 → Gly (+ Δ 88 Da and + Δ 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive β-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Δ 70 Da) and a hydrated aldehyde (+ Δ 88 Da) as stable products of inhibition. Our results reveal that the Ser → Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser130 → Gly β-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser70 in this enzyme. After acylation of tazobactam by Ser130 → Gly, inactivation proceeds independent of any additional covalent interactions. β-Lactam antibiotics are the most frequently prescribed antibacterial agents used to treat infections. This class of antibiotics enjoys target specificity (bacterial cell wall synthesizing transpeptidases and carboxypeptidases) and safety. β-Lactamases (EC 3.5.2.6) threaten the continued efficacy of β-lactams. These ubiquitous enzymes are produced by a wide variety of microorganisms including anaerobic and aerobic Gram-positive and Gram-negative bacteria. Currently, there are nearly 470 β-lactamases assembled into four distinct molecular classes (classes A–D) or groups (Bush-Jacoby-Mederios Group 1–4) (1Bush K. Clin. Infect. Dis. 2001; 32: 1085-1089Crossref PubMed Scopus (358) Google Scholar, 2Bush K. Curr. Opin. Investig. Drugs. 2002; 3: 1284-1290PubMed Google Scholar, 3Bush K. Jacoby G.A. Medeiros A.A. Antimicrob. Agents Chemother. 1995; 39: 1211-1233Crossref PubMed Scopus (2155) Google Scholar, www.lahey.org/studies/webt.asp). Classes A, C, and D are serine-reactive hydrolases that inactivate the β-lactam by acylation of the active site serine followed by hydrolysis of the acyl-enzyme intermediate. The class B enzymes are zinc-dependent hydrolases (3Bush K. Jacoby G.A. Medeiros A.A. Antimicrob. Agents Chemother. 1995; 39: 1211-1233Crossref PubMed Scopus (2155) Google Scholar, 4Galleni M. Lamotte-Brasseur J. Rossolini G.M. Spencer J. Dideberg O. Frere J.M. Antimicrob. Agents Chemother. 2001; 45: 660-663Crossref PubMed Scopus (338) Google Scholar, 5Helfand M.S. Bonomo R.A. Curr. Drug Targets Infect. Disord. 2003; 3: 9-23Crossref PubMed Scopus (80) Google Scholar). The increasing number of serine and metallo-β-lactamases with extended substrate specificity is seriously threatening the ability of clinicians to treat infections in the hospital, nursing home, and community settings. An urgent need exists to develop novel β-lactams. One of the most successful strategies used to overcome β-lactamase-mediated resistance is the use of β-lactamase inhibitors (6Page M.G. Drug Resist. Updat. 2000; 3: 109-125Crossref PubMed Scopus (72) Google Scholar). These mechanism-based inhibitors act by covalently modifying the active site of class A β-lactamases. Tazobactam, sulbactam, and clavulanic acid are the current β-lactamase inhibitors used in clinical practice (Fig. 1). Each is coupled with a β-lactam antibiotic to improve the potency and longevity of the partner drug. Commercial preparations of β-lactam β-lactamase inhibitors available in the United States include piperacillin/tazobactam (Zosyn™), ampicillin/sulbactam (Unasyn™), amoxicillin/clavulanate (Augmentin™), and ticarcillin/clavulanate (Timentin™). As a group, these β-lactamase inhibitors are primarily effective against susceptible class A β-lactamases. Unfortunately, single amino acid substitutions in the class A TEM and SHV families of β-lactamases have resulted in enzymes with reduced affinity for β-lactamase inhibitors. TEM and SHV β-lactamases that are resistant to inhibitors have alterations at Ambler positions Met69, Ser130, Arg244, Arg275, and Asp276 (7Ambler R.P. Coulson A.F. Frere J.M. Ghuysen J.M. Joris B. Forsman M. Levesque R.C. Tiraby G. Waley S.G. Biochem. J. 1991; 276: 269-272Crossref PubMed Scopus (871) Google Scholar, 8Hujer A.M. Hujer K.M. Bonomo R.A. Biochim. Biophys. Acta. 2001; 1547: 37-51Crossref PubMed Scopus (62) Google Scholar, 9Vakulenko S.B. Geryk B. Kotra L.P. Mobashery S. Lerner S.A. Antimicrob. Agents Chemother. 1998; 42: 1542-1548Crossref PubMed Google Scholar, 10Helfand M.S. Hujer A.M. Sonnichsen F.D. Bonomo R.A. J. Biol. Chem. 2002; 277: 47719-47723Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Alonso R. Gerbaud G. Galimand M. Courvalin P. Antimicrob. Agents Chemother. 2002; 46: 3627-3629Crossref PubMed Scopus (6) Google Scholar, 12Canica M.M. Barthelemy M. Gilly L. Labia R. Krishnamoorthy R. Paul G. Antimicrob. Agents Chemother. 1997; 41: 374-378Crossref PubMed Google Scholar) (www.lahey.org/studies/webt.asp). The recent crystal structure determinations of inhibitor-resistant TEMs and SHV-1 β-lactamases help explain why resistance to clavulanic acid and tazobactam is observed (13Meroueh S.O. Roblin P. Golemi D. Maveyraud L. Vakulenko S.B. Zhang Y. Samama J.P. Mobashery S. J. Am. Chem. Soc. 2002; 124: 9422-9430Crossref PubMed Scopus (47) Google Scholar, 14Swaren P. Golemi D. Cabantous S. Bulychev A. Maveyraud L. Mobashery S. Samama J.P. Biochemistry. 1999; 38: 9570-9576Crossref PubMed Scopus (45) Google Scholar, 15Wang X. Minasov G. Shoichet B.K. J. Biol. Chem. 2002; 277: 32149-32156Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16Kuzin A.P. Nukaga M. Nukaga Y. Hujer A.M. Bonomo R.A. Knox J.R. Biochemistry. 1999; 38: 5720-5727Crossref PubMed Scopus (113) Google Scholar, 17Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar). Subtle changes in the molecular dynamics of the Ω loop region (Ambler positions 145–179 and 155–162) affecting the dissociation constant (KI) yield the inhibitor-resistant phenotype in the Met69 → Leu amino acid substitution of TEM (13Meroueh S.O. Roblin P. Golemi D. Maveyraud L. Vakulenko S.B. Zhang Y. Samama J.P. Mobashery S. J. Am. Chem. Soc. 2002; 124: 9422-9430Crossref PubMed Scopus (47) Google Scholar). For Asn276 → Asp variant of TEM, the formation of a salt bridge from Asp to the side chain of Arg244 and loss of a bridging water molecule lie at the heart of the resistance for this TEM to clavulanic acid inactivation (14Swaren P. Golemi D. Cabantous S. Bulychev A. Maveyraud L. Mobashery S. Samama J.P. Biochemistry. 1999; 38: 9570-9576Crossref PubMed Scopus (45) Google Scholar). Movement of Ser130 in the inhibitor-resistant TEM-30 (Arg244 → Ser), TEM-32 (Met69 → Ile, Met182 → Thr), and TEM-34 (Met69 → Val) β-lactamases emphasizes the strategic role of Ser130 (15Wang X. Minasov G. Shoichet B.K. J. Biol. Chem. 2002; 277: 32149-32156Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In addition, Kuzin et al. (16Kuzin A.P. Nukaga M. Nukaga Y. Hujer A.M. Bonomo R.A. Knox J.R. Biochemistry. 1999; 38: 5720-5727Crossref PubMed Scopus (113) Google Scholar, 17Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar) further illustrated the importance of Ser130 in their structural analysis of the SHV-1 and tazobactam-inactivated SHV-1. The crystal structure of the SHV-1-tazobactam complex showed the formation of an acyclic form of tazobactam attached to Ser70 (assigned to the imine on the basis of dihedral angles) and a 5-atom vinyl carboxylic acid fragment attached to Ser130-OH in the inactive species. Electrospray ionization mass spectrometry (ESI/MS) 1The abbreviations used are: LC-ESI/MS, liquid chromatography-electrospray ionization/mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight mass spectrometry; HPLC, high pressure liquid chromatography. is becoming an essential tool in determining the covalent intermediates of hydrolysis and inactivation by serine β-lactamases (18Saves I. Burlet-Schiltz O. Maveyraud L. Samama J.P. Prome J.C. Masson J.M. Biochemistry. 1995; 34: 11660-11667Crossref PubMed Scopus (52) Google Scholar, 19Aplin R.T. Baldwin J.E. Schofield C.J. Waley S.G. FEBS Lett. 1990; 277: 212-214Crossref PubMed Scopus (47) Google Scholar, 20Zervosen A. Valladares M.H. Devreese B. Prosperi-Meys C. Adolph H.W. Mercuri P.S. Vanhove M. Amicosante G. van Beeumen J. Frere J.M. Galleni M. Eur. J. Biochem. 2001; 268: 3840-3850Crossref PubMed Scopus (30) Google Scholar, 21Payne D.J. Bateson J.H. Tolson D. Gasson B. Khushi T. Ledent P. Frere J.M. Biochem. J. 1996; 314: 457-461Crossref PubMed Scopus (10) Google Scholar, 22Brown R.P. Aplin R.T. Schofield C.J. Biochemistry. 1996; 24: 12421-12432Crossref Scopus (101) Google Scholar, 23Saves I. Burlet-Schiltz O. Swaren P. Lefevre F. Masson J.M. Prome J.C. Samama J.P. J. Biol. Chem. 1995; 270: 18240-18245Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 24Yang Y. Janota K. Tabei K. Huang N. Siegel M.M. Lin Y.I. Rasmussen B.A. Shlaes D.M. J. Biol. Chem. 2000; 275: 26674-26682Abstract Full Text Full Text PDF PubMed Google Scholar). The role of Asn276 → Asp in the inhibitor-resistant TEM-35 and TEM-36 (inhibitor-resistant TEM-4 and -7) was first examined by ESI/MS (23Saves I. Burlet-Schiltz O. Swaren P. Lefevre F. Masson J.M. Prome J.C. Samama J.P. J. Biol. Chem. 1995; 270: 18240-18245Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Saves et al. (18Saves I. Burlet-Schiltz O. Maveyraud L. Samama J.P. Prome J.C. Masson J.M. Biochemistry. 1995; 34: 11660-11667Crossref PubMed Scopus (52) Google Scholar) showed a cleaved moiety of clavulanic acid leads to the formation of the major inactive product; the mass of the clavulanate-Asn276 → Asp complex was 80 Da greater than the mass of the uninhibited enzyme. By studying clavulanic acid inhibition of TEM-2 β-lactamase, four different mass increments relative to the uninhibited TEM-2 were found (+ Δ 52, 70, 88, and 155 Da) (22Brown R.P. Aplin R.T. Schofield C.J. Biochemistry. 1996; 24: 12421-12432Crossref Scopus (101) Google Scholar). The Ser70 and Ser130 amino acids were identified as modified residues in the inactivation process. Extending this work, Yang et al. (24Yang Y. Janota K. Tabei K. Huang N. Siegel M.M. Lin Y.I. Rasmussen B.A. Shlaes D.M. J. Biol. Chem. 2000; 275: 26674-26682Abstract Full Text Full Text PDF PubMed Google Scholar) demonstrated that the inactivation of PC 1 and TEM-1 β-lactamases by tazobactam results in four additions to PC 1 β-lactamase (+ Δ 52, 70, 88, and 300 Da) and three additions to TEM-1 β-lactamase (+ Δ 52, 70, and 88 Da). As in the case with clavulanic acid inactivation of TEM-2, the amino acids Ser70 and Ser130 were again the tazobactam-modified residues. Evidence for an aldehyde and hydrated aldehyde attached to Ser70 was found. A 70-Da increase in mass attached to the Ser130 residue was also detected. Hence, the critical involvement of Ser130 in the inactivation process, as first emphasized by Imitiaz et al. (25Imtiaz U. Billings E.M. Knox J.R. Mobashery S. Biochemistry. 1994; 33: 5728-5738Crossref PubMed Scopus (76) Google Scholar), is central to the studies of the inhibition of class A β-lactamases by clavulanic acid and tazobactam. Investigations by Helfand et al. (26Helfand M.S. Bethel C.R. Hujer A.M. Hujer K.M. Anderson V.E. Bonomo R.A. J. Biol. Chem. 2003; 278: 52724-52729Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) stressed that the relative differences in kinact values between SHV-1 and Ser130 → Gly, an inhibitor-resistant variant, were smaller than the differences in the dissociation constant for the pre-acylation complex, KI, for tazobactam and clavulanic acid. Furthermore, the apparent inhibitor to enzyme (I/E) ratios required to inactivate SHV-1 and Ser130 → Gly β-lactamases were similar; the inactivation process is hindered only by the affinities of clavulanic acid and tazobactam for the active site of the Ser130 → Gly variant. These observations led us to investigate the nature of the inhibited intermediates when Gly is substituted for Ser at position 130. Here we report the study of the inhibition reaction of SHV-1 and Ser130 → Gly-substituted SHV-1 β-lactamases with tazobactam by using LC-ESI/MS to determine the mass increase after the inhibition. Tryptic digests were analyzed using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Examining the inhibitor-resistant Ser130 → Gly β-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser70 in this enzyme. After acylation of tazobactam by Ser130 → Gly, we propose that inactivation proceeds independent of any additional modifications. Preparation of SHV-1 and Ser130 → Gly-substituted β-Lactamases— The genetic constructs and Escherichia coli DH10B strains harboring the SHV-1 and Ser130 → Gly β-lactamases were described previously (8Hujer A.M. Hujer K.M. Bonomo R.A. Biochim. Biophys. Acta. 2001; 1547: 37-51Crossref PubMed Scopus (62) Google Scholar). The mature SHV-1 β-lactamase is a 265-amino acid protein with a calculated Mr 28,874 (16Kuzin A.P. Nukaga M. Nukaga Y. Hujer A.M. Bonomo R.A. Knox J.R. Biochemistry. 1999; 38: 5720-5727Crossref PubMed Scopus (113) Google Scholar). Ser130 → Gly was first constructed using site-directed mutagenesis (8Hujer A.M. Hujer K.M. Bonomo R.A. Biochim. Biophys. Acta. 2001; 1547: 37-51Crossref PubMed Scopus (62) Google Scholar, 10Helfand M.S. Hujer A.M. Sonnichsen F.D. Bonomo R.A. J. Biol. Chem. 2002; 277: 47719-47723Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). SHV-1 and Ser130 → Gly β-lactamases were purified to greater than 95% homogeneity by preparative isoelectric focusing according to established procedures in our laboratory (10Helfand M.S. Hujer A.M. Sonnichsen F.D. Bonomo R.A. J. Biol. Chem. 2002; 277: 47719-47723Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 26Helfand M.S. Bethel C.R. Hujer A.M. Hujer K.M. Anderson V.E. Bonomo R.A. J. Biol. Chem. 2003; 278: 52724-52729Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The eluting and storage buffers for SHV-1 and Ser130 → Gly β-lactamases were 20 mm diethanolamine, pH 8.5. Inactivation of SHV-1 and Ser130 → Gly-substituted β-Lactamases by Tazobactam—SHV-1 β-lactamase (76 μm) and Ser130 → Gly-substituted SHV-1 β-lactamase (38 μm) stock solutions were used in these experiments. Sodium tazobactam (molecular mass 330 Da) was a kind gift from Wyeth Pharmaceuticals (Pearl River, NY). Tazobactam was dissolved in HPLC grade water and mixed with SHV-1 and with the Ser130 → Gly β-lactamase in a molar ratio of 1000:1 (inhibitor/enzyme) at room temperature for 30 min. Immediately following the reaction, the inhibited enzymes were purified and characterized by the LC-MS procedure described below. All protein analyses were performed in HPLC grade water. LC-ESI/MS—The LC-ESI/MS analysis of all samples was performed using a Micromass (Beverly, MA) Quattro II triple quadrupole mass spectrometer with electrospray ionization interfaced to an Agilent (Colorado Springs, CO) 1100 series HPLC system as described previously (27Bonomo R.A. Liu J. Chen Y. Ng L. Hujer A.M. Anderson V.E. Biochim. Biophys. Acta. 2001; 1547: 196-205Crossref PubMed Scopus (25) Google Scholar). The SHV-1, Ser130 → Gly β-lactamase, tazobactam-inactivated SHV-1, and the Ser130 → Gly enzymes were first separated on a C4 reverse phase column (1 × 150 mm, 5 μm, 300 Å) from Vydac (Hesperia, CA). The mobile phase is a gradient mixture of A, 0.3% acetic acid in water, and B, 0.3% acetic acid in acetonitrile with a flow rate through the column of 45 μl/min. The gradient timetable was 2% B at 0 min and 80% B at 30 min, and the total run time was 60 min. The parameters for the ESI/MS are as follows: ionization mode was positive; cone voltage was 70 V; nitrogen was used both as a nebulizing and as a drying gas; the source temperature was 70 °C; capillary voltage was 3.5 kV; the spectra were obtained scanning over 800–2400 Da. Proteolysis—Sequencing grade trypsin (Promega, Madison, WI) was used to digest both uninhibited and tazobactam-inhibited SHV-1 and the Ser130 → Gly β-lactamases. The uninhibited and inactivated β-lactamases were denatured with 8 m urea at 50 °C for 30 min. The pH of the denaturation mixture was adjusted to 8 using 0.4 m NH4HCO3. After a 1:4 dilution with water, trypsin was added into the mixture at a β-lactamase/trypsin ratio of 25:1 (w/w) and incubated at 37 °C for 12 h. Freezing the mixture at –40 °C terminated proteolysis. The digestion products were analyzed by LC-ESI/MS and by MALDI-TOF MS using the conditions described below. LC-ESI/MS for the Tryptic Digests—The tryptic digests of the uninhibited and tazobactam-inactivated β-lactamases were separated on a C18 column (1 × 150 mm, 5 μm, 300 Å) (Vydac). The mobile phase was a gradient mixture of A, 0.3% acetic acid in water, and B, 0.3% acetic acid in acetonitrile from 2 to 80% B in 70 min with a flow rate through the column of 35 μl/min. The total run time was 120 min. The ESI/MS parameters were the same as described above, except that cone voltage was 45 V and the scanning range was 300–1700 Da. MALDI-TOF MS of the Tryptic Digests—The tryptic digests were analyzed using an Omniflex MALDI-TOF Mass Spectrometer from Bruker (Billerica, MA). Positive ionization and reflectron mode were used. The experimental parameters are as follows: laser power 60%, voltage 76.4%, and ion focus 9.2. The matrix was α-cyano-4-hydroxycinnamic acid. The internal standard consisted of bradykinin, renin substrate, and adrenocorticoid hormone fragments 18–39 and 7–38 (Sigma). The samples were de-salted using C18 Zip-Tips from Millipore (Bedford, MA) before mixing with the matrix. LC-ESI/MS, MALDI-TOF MS, and the Inhibition of SHV-1 β-Lactamase—SHV-1 and tazobactam-inhibited SHV-1 β-lactamase were first separated and characterized using LC-ESI/MS. The scan mass spectra and the deconvoluted spectra for the chromatographic peaks of the enzymes are shown in Fig. 2. As can be seen, the molecular mass of the apoenzyme and tazobactam-inhibited SHV-1 β-lactamases measured by ESI/MS are 28,872 ± 3 and 28,959 ± 3 Da, respectively. This represents a mass increase of 87 ± 6 Da after inhibition. This additional mass is lower than the molecular mass of tazobactam indicating that a cleaved moiety of the inhibitor is covalently attached in the tazobactam-SHV-1 complex. The mass increment is consistent with the 88-Da increase seen in the tazobactam inactivation of PC 1 and TEM-1 and clavulanic acid inhibition of TEM-2 (22Brown R.P. Aplin R.T. Schofield C.J. Biochemistry. 1996; 24: 12421-12432Crossref Scopus (101) Google Scholar, 24Yang Y. Janota K. Tabei K. Huang N. Siegel M.M. Lin Y.I. Rasmussen B.A. Shlaes D.M. J. Biol. Chem. 2000; 275: 26674-26682Abstract Full Text Full Text PDF PubMed Google Scholar). To identify the amino acid site of attachment, the intact and inhibited SHV-1 were digested with trypsin, and the tryptic digests were analyzed by LC-ESI/MS and MALDI-TOF MS. Twenty four peptides were identified by LC-ESI/MS from the tryptic digest, and 18 peptides were assigned by MALDI-TOF MS (Table I). We observed from the LC-ESI/MS data that the relative intensity of peptide 11 containing residues 66–73 (987.5 Da) and peptide 20 containing residues 62–73 (1458.7 Da) was dramatically decreased after tazobactam inhibition. As shown in Figs. 3A and 4A, the MALDI-TOF spectrum for the tryptic digest of the uninhibited SHV-1 had signals from the [M + H] ions of the 988.46 and 1459.7, respectively. However, the tryptic digest of the tazobactam-inhibited SHV-1 did not show the peaks for those peptides (Figs. 3B and 4B). These data indicate that the modification site is located between residues 66 and 73 of the SHV-1 β-lactamase. Hence, we demonstrate that a stable intermediate of inhibition of SHV-1 β-lactamase with tazobactam involves attachment of a fragment of this inhibitor to an amino acid segment containing Ser70. Based upon previous structural and ESI/MS studies, it is unlikely that Lys73 is the modified residue (17Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar, 25Imtiaz U. Billings E.M. Knox J.R. Mobashery S. Biochemistry. 1994; 33: 5728-5738Crossref PubMed Scopus (76) Google Scholar).Table ISHV-1 and tazobactam-inhibited SHV-1 β -lactamase peptides identified with LC-ESI/MS and MALDI-TOF/MSPeptides identified by LC-ESI/MSMass by LC-ESI/MSAmino acid residue assignmentsPeptide identified by LC-ESI/MS[M + H]Peptide identified by MALDI-TOF/MSUninhibited SHV-1Tazobactam-inhibited SHV-1SHV-1Tazobactam-inactivated SHV-11489.262-65++--2715.494-98++++3716.4193-198++717.42++4724.5216-222++725.47++5746.456-61++747.41++6844.5192-198++--7874.6258-264++875.57++8901.5154-161++902.46--9975.535-43++976.50--10976.4265-273++977.44++11987.566-73+-988.46+-121038.625-34++1039.58++131094.6244-254++1095.65++141130.584-93++1131.53++151277.644-55++1278.61++161285.7153-164++++171302.6206-215++1303.64--181302.7223-234++1303.70++191334.7179-191++1335.65++201458.762-73+-1459.70+-211506.799-111++1507.72++221599.8165-178++1600.76--231904.0274-290++1095.01++241984.0162-178++++a112-1534114.08b235-40590.28c95-98588.32d74-831000.60 Open table in a new tab Fig. 4Zoomed MALDI-TOF spectra for the tryptic digests of SHV-1 (A) and the tazobactam-inhibited SHV-1 (B). *, note absent peptide corresponding to amino acids 62–73 (1459.7).View Large Image Figure ViewerDownload (PPT) A new peak was observed at m/z 1076.3 in the MALDI-TOF mass spectrum for the digest of the inhibited SHV-1 (Fig. 3B). The mass difference between this new peak and the disappeared peptide peak at 988.5 m/z (residues 66–72) is 87.8 Da and is in excellent agreement with the mass increase of 87 ± 6 Da for SHV-1 after inhibition. This new peak with an m/z of 1076.3 establishes that a moiety with a mass of 88 Da is attached to the peptide with m/z 988.5. LC-ESI/MS, MALDI-TOF MS, and the Inhibition of Ser130 → Gly β-Lactamase—Ser130 → Gly and tazobactam-inhibited Ser130 → Gly variants of SHV-1 β-lactamase were studied in the same manner as the SHV-1 enzyme. By using LC-ESI/MS, we determined that the Ser130 → Gly β-lactamase had a mass increase of 70 ± 6 Da from 28,839 ± 3 to 28,909 ± 3 Da (Fig. 5) after inhibition with tazobactam. The analyses of the tryptic digests of the Ser130 → Gly β-lactamase and inhibited Ser130 → Gly β-lactamase by LC-ESI/MS and MALDI-TOF MS are presented in Figs. 6 and 7 and listed in Table II. As shown, the peptides with m/z 988.3 (residues 66–73) and 1459.7 (residues 62–73) by MALDI-TOF MS are again present in the digest of the Ser130 → Gly-substituted protein but have largely disappeared in the digest of the tazobactam-inhibited Ser130 → Gly β-lactamase. The results show that a fragment of tazobactam is still attached to Ser70 in Ser130 → Gly β-lactamase and that Ser130 → Gly and SHV-1 β-lactamases follow similar pathways to inactivation.Fig. 7MALDI-TOF spectra for the tryptic digests of Ser130 → Gly (A) and the tazobactam-inhibited Ser130 → Gly (B). Arrow indicates loss of a peptide peak at mass 1459.6.View Large Image Figure ViewerDownload (PPT)Table IISer130 → Gly β -lactamase and tazobactam-inhibited Ser130 → Gly β -lactamase peptides identified with LC-ESI/MS and MALDI-TOF/MSPeptides identified by LC-ESI/MSMass by LC-ESI/MS (±3)Amino acid residue assignmentsPeptide identified[M + H]Peptide identified by MALDI-TOF/MSSHV-1Tazobactam-inhibited SHV-1SHV-1Tazobactam-inactivated SHV-11489.262-65++--2715.494-98++++3716.4193-198++717.42++4724.5216-222++725.47++5746.456-61++747.41++6844.5192-198++--7874.6258-264++875.57++8901.5154-161++902.46--9975.535-43++976.50--10976.4265-273++977.44++11987.566-73+-988.46+-121038.625-34++1039.58++131094.6244-254++1095.65++141130.584-93++1131.53++151277.644-55++1278.61++161285.7153-164++++171302.6206-215++1303.64--181302.7223-234++1303.70++191334.7179-191++1335.65++201458.762-73--+-211506.799-111++1507.72++221599.8165-178++1600.76--231904.0274-290++1095.00++241984.0162-178++++a112-1534114.08b235-40590.28c95-98588.32d74-831000.60 Open table in a new tab Based on the ratio of tazobactam/β-lactamase and the time for inactivation (30 min), a mechanism explaining the inhibition of Ser130 → Gly β-lactamase by tazobactam is proposed (Fig. 8). In this scheme, the Ser70-OH of Ser130 → Gly β-lactamase attacks the carbon of the carbonyl group in the β-lactam ring 1. This leads to the acyl-enzyme 2 that undergoes further reaction to generate a linear imine species 3 after thiazolidine ring opening and departure of the sulfinate from C-5. This linear imine leads to an enamine 4 via rearrangement. Addition of a water molecule to the imine 3 and/or enamine 4 yields an aldehyde 5 that has a 70-Da mass increase. For SHV-1, the hydration of the aldehyde 5 leads to the product 6 with an 88-Da mass increase. This pathway is consistent with our mass spectrometry studies, with detailed kinetic analysis of the Ser130 → Gly mutant of SHV-1 and other inhibitor-resistant enzymes, and is reminiscent of the reaction of CMY-2 β-lactamase with tazobactam (14Swaren P. Golemi D. Cabantous S. Bulychev A. Maveyraud L. Mobashery S. Samama J.P. Biochemistry. 1999; 38: 9570-9576Crossref PubMed Scopus (45) Google Scholar, 15Wang X. Minasov G. Shoichet B.K. J. Biol. Chem. 2002; 277: 32149-32156Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 17Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar, 22Brown R.P. Aplin R.T. Schofield C.J. Biochemistry. 1996; 24: 12421-12432Crossref Scopus (101) Google Scholar, 24Yang Y. Janota K. Tabei K. Huang N. Siegel M.M. Lin Y.I. Rasmussen B.A. Shlaes D.M. J. Biol. Chem. 2000; 275: 26674-26682Abstract Full Text Full Text PDF PubMed Google Scholar). We propose that this reaction mechanism is independent of any additional covalent interactions with the inhibitor-resistant β-lactamase, except for the attachment to Ser70. It is also remarkable that we have not found a mass consistent with formation of the acyl-enzyme species. This observation adds significant importance to the chemical properties of tazobactam interacting with the SHV β-lactamase. Most notably, our results reveal that the Ser → Gly substitution at amino acid position 130 is not essential for enzyme inactivation, and the five atom vinyl carboxylic acid species is not the exclusive terminal species of inhibition in the SHV-1 or Ser130 → Gly β-lactamase (17Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar). The absence of a Ser-OH at position 130 could affect the process of proton shuttling. Studying the inhibition of SHV-1 β-lactamase with tazobactam by using x-ray crystallography, Kuzin et al. (17Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar) demonstrated that in the formation of the final inactivation product, the hydroxyl group of Ser130 acts as a nucleophile to react with an intermediate (imine) of tazobactam covalently attached to Ser70. As a result, the tazobactam remnant is attached to Ser130 yielding an inactive enzyme. The absence of the Ser130-OH deprives the altered enzyme of the nucleophile believed to act in the final irreversible inactivation step (15Wang X. Minasov G. Shoichet B.K. J. Biol. Chem. 2002; 277: 32149-32156Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 17Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar, 24Yang Y. Janota K. Tabei K. Huang N. Siegel M.M. Lin Y.I. Rasmussen B.A. Shlaes D.M. J. Biol. Chem. 2000; 275: 26674-26682Abstract Full Text Full Text PDF PubMed Google Scholar). Nevertheless, a pathway for tazobactam inhibition of Ser130 → Gly enzyme involving a modification of tazobactam while still attached to Ser70 exists (26Helfand M.S. Bethel C.R. Hujer A.M. Hujer K.M. Anderson V.E. Bonomo R.A. J. Biol. Chem. 2003; 278: 52724-52729Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). As stated in our kinetic analysis of the Ser130 → Gly-substituted β-lactamase, we favor the hypothesis that movement or displacement of an active site water molecule may facilitate this particular reaction pathway (26Helfand M.S. Bethel C.R. Hujer A.M. Hujer K.M. Anderson V.E. Bonomo R.A. J. Biol. Chem. 2003; 278: 52724-52729Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Alternatively, the presence of a residue on a neighboring amino acid may also serve as the reactive nucleophile. Our data do not permit us to favor one explanation over another. However, we did not see any modification of the peptide containing Lys234, the likely candidate to assume the role of Ser130 (17Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar, 25Imtiaz U. Billings E.M. Knox J.R. Mobashery S. Biochemistry. 1994; 33: 5728-5738Crossref PubMed Scopus (76) Google Scholar). Finally, it is puzzling that we consistently see the formation of the aldehyde (+ Δ70) in the Ser130 → Gly-tazobactam inactivated enzyme and observe the hydrated aldehyde (+Δ88) in the SHV-1-tazobactam inactivated enzyme. This finding may be an important clue revealing differences in the strategic location of water molecules in the active site because of the Ser130 → Gly substitution. By identifying stable inactivation products in both SHV-1 and Ser130 → Gly β-lactamases, our studies emphasize that unique reaction chemistry is undergone by mechanism-based inhibitors interacting with "inhibitor-resistant" β-lactamases. It has not escaped our attention that some evidence is present for the formation of more than a single inhibited species. Close analysis of Figs. 2 and 5 reveals smaller peaks adjacent to the predominant species identified by ESI/MS. The relative size of these peaks may indicate the proportion of the intermediates formed. We chose to target our analysis on the predominant species. Studies probing the mechanism of inactivation of inhibitor-resistant β-lactamases employing x-ray crystallography and other spectroscopic modalities will undoubtedly shed further light upon the interactions between β-lactamase inhibitors and inhibitor-resistant β-lactamases and lead to the design of more effective therapy (28Helfand M.S. Totir M.A. Carey M.P. Hujer A.M. Bonomo R.A. Carey P.R. Biochemistry. 2003; 42: 13386-13392Crossref PubMed Scopus (64) Google Scholar). We thank Yindin Liu and Deley Sulton for their technical assistance and Dr. James R. Knox for helpful discussions and a critical review of this manuscript.

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