Mechanism of Inhibition of the Class A β-Lactamases PC1 and TEM-1 by Tazobactam (2000)

The reactions of class A β-lactamases PC1 and TEM-1 with tazobactam (TZB), a potent penicillanic sulfone inhibitor for class A β-lactamases, were studied using electrospray ionization mass spectrometry (ESI/MS). Following inactivation of the β-lactamases by TZB, new abundant high mass components were observed including three with molecular masses of 52, 70, and 88 Da greater than PC1 and TEM-1, respectively, and a component with a molecular mass of 300 Da greater than PC1. In addition, three TZB reaction products with molecular masses of 248, 264, and 280 Da were observed. High performance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct digested with Glu-C revealed three new components with masses 52, 70, and 88 Da greater than that of the peptide composed of amino acid residues 58–82 and one new component with a mass 70 Da greater than that of the peptide composed of amino acid residues 125–141. HPLC/ESI/MS/MS analysis of the two digested peptides whose masses increased by 70 Da indicated that Ser-70 and Ser-130 were the most likely TZB-modified amino acid residues. Based on these data, a mechanism for the inactivation of the class A β-lactamases by TZB is proposed. In this scheme, initial acylation of Ser-70 by TZB and opening of the lactam ring are followed by one of several different events: (1Bonomo R.A Rudin S.E.A. Shlaes D.M. FEMS Microbiol. Lett. 1997; 148: 59-62Crossref PubMed Scopus (34) Google Scholar) the rapid decomposition of TZB with loss of the enamine moiety to form the propiolylated enzyme, (2Payne D.J. Cramp R. Winstanley D.J. Knowles D.J.C. Antimicrob. Agents Chemother. 1994; 38: 767-772Crossref PubMed Scopus (147) Google Scholar) an intramolecular nucleophilic displacement of the imine or enamine moiety by Ser-130 to form a cross-linked vinyl ether, and (3Kuck N.A. Jacobus N.V. Petersen P.J. Weiss W.J. Testa R.T. Antimicrob. Agents Chemother. 1989; 33: 1964-1969Crossref PubMed Scopus (86) Google Scholar) hydrolysis of the imine or enamines to form a Ser-70-linked aldehyde. The reactions of class A β-lactamases PC1 and TEM-1 with tazobactam (TZB), a potent penicillanic sulfone inhibitor for class A β-lactamases, were studied using electrospray ionization mass spectrometry (ESI/MS). Following inactivation of the β-lactamases by TZB, new abundant high mass components were observed including three with molecular masses of 52, 70, and 88 Da greater than PC1 and TEM-1, respectively, and a component with a molecular mass of 300 Da greater than PC1. In addition, three TZB reaction products with molecular masses of 248, 264, and 280 Da were observed. High performance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct digested with Glu-C revealed three new components with masses 52, 70, and 88 Da greater than that of the peptide composed of amino acid residues 58–82 and one new component with a mass 70 Da greater than that of the peptide composed of amino acid residues 125–141. HPLC/ESI/MS/MS analysis of the two digested peptides whose masses increased by 70 Da indicated that Ser-70 and Ser-130 were the most likely TZB-modified amino acid residues. Based on these data, a mechanism for the inactivation of the class A β-lactamases by TZB is proposed. In this scheme, initial acylation of Ser-70 by TZB and opening of the lactam ring are followed by one of several different events: (1Bonomo R.A Rudin S.E.A. Shlaes D.M. FEMS Microbiol. Lett. 1997; 148: 59-62Crossref PubMed Scopus (34) Google Scholar) the rapid decomposition of TZB with loss of the enamine moiety to form the propiolylated enzyme, (2Payne D.J. Cramp R. Winstanley D.J. Knowles D.J.C. Antimicrob. Agents Chemother. 1994; 38: 767-772Crossref PubMed Scopus (147) Google Scholar) an intramolecular nucleophilic displacement of the imine or enamine moiety by Ser-130 to form a cross-linked vinyl ether, and (3Kuck N.A. Jacobus N.V. Petersen P.J. Weiss W.J. Testa R.T. Antimicrob. Agents Chemother. 1989; 33: 1964-1969Crossref PubMed Scopus (86) Google Scholar) hydrolysis of the imine or enamines to form a Ser-70-linked aldehyde. clavulanic acid tazobactam electrospray ionization mass spectrometry high performance liquid chromatography molecular weight fast atom bombardment tazobactam and PC1 covalent adduct tazobactam and TEM-1 covalent adduct acetonitrile Two structurally distinct classes of β-lactamase inhibitors, clavams represented by clavulanic acid (CA,1 see Structure Fs1) and penicillanic sulfones represented by sulbactam and tazobactam (TZB, see Structure Fs1), have been widely used clinically. In combination with a β-lactam antibiotic, these inhibitors have successfully overcome bacterial β-lactam resistance caused by β-lactamase-mediated β-lactam hydrolysis. In particular, tazobactam, a triazoly-substituted penicillanic sulfone, has potent inhibitory activity against class A β-lactamases, including some β-lactamases that are resistant to inactivation by CA and sulbactam (1Bonomo R.A Rudin S.E.A. Shlaes D.M. FEMS Microbiol. Lett. 1997; 148: 59-62Crossref PubMed Scopus (34) Google Scholar, 2Payne D.J. Cramp R. Winstanley D.J. Knowles D.J.C. Antimicrob. Agents Chemother. 1994; 38: 767-772Crossref PubMed Scopus (147) Google Scholar). Extensive studies have demonstrated that the combination of tazobactam-piperacillin is an effective antimicrobiological agent against class A β-lactamase producing isolates (3Kuck N.A. Jacobus N.V. Petersen P.J. Weiss W.J. Testa R.T. Antimicrob. Agents Chemother. 1989; 33: 1964-1969Crossref PubMed Scopus (86) Google Scholar, 4Murray P.R. Cantrell H.F. Lankford R.B. Diagn. Microbiol. Infect. Dis. 1994; 19 (the In Vitro Susceptibility Surveillance Group): 111-120Crossref PubMed Scopus (55) Google Scholar). However, direct evidence of the mechanism of inactivation of the class A β-lactamases by the penicillanic sulfone inhibitors has not been fully addressed. The mechanism of β-lactamase inactivation by CA has been studied extensively. Crystallographic studies performed on the clavulanic acid and PC1 covalent adduct revealed the structure of the acyl-enzyme complex to consist of a CA fragment covalently bound to the active site Ser-70, as either the cis or trans decarboxylated enamines (5Chen C.C. Herzberg O. J. Mol. Bacteriol. 1992; 224: 1103-1113Google Scholar). ESI/MS studies by Brown et al. (6Brown R.P. A Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar) provided direct evidence for the complexation of CA with the TEM-2 β-lactamase. CA fragments with masses of 70 and 88 Da were attached to the peptide fragment containing Ser-70, and a second 70-Da mass fragment, assigned as a β-linked acrylate, was localized to a peptide containing Ser-130. The mechanism of inactivation of TZB for all major classes of β-lactamases has been studied using UV spectrometric assays (7Bush K. Macalintal C. Rasmussen B.A. Lee V. Yang Y. Antimicrob. Agents Chemother. 1993; 37: 851-858Crossref PubMed Scopus (235) Google Scholar). The formation of a transient complex and generation of subsequent degradation products were observed. For the class A β-lactamases PC1 and TEM-2, a new chromophore absorbing at 288 nm indicated the formation of reaction intermediates. It was proposed that these intermediates were the products formed following deacylation. An analysis of the mechanism of inactivation of class A β-lactamases by CA and sulbactam (8Imtiaz U. Billings E.M. Knox J.R. Mobashery S. Biochemistry. 1994; 33: 5728-5738Crossref PubMed Scopus (76) Google Scholar) indicated that the main differences in β-lactamase inactivation by CA and sulbactam are in the events leading to β-elimination and opening of the five-membered ring. For CA, efficient β-elimination occurs following protonation. This proton is donated by an active site water molecule (9Imtiaz U. Billings E.M. Knox J.R. Manavathu E., K. Lerner S.A. Mobashery S. J. Am. Chem. Soc. 1993; 115: 4435-4442Crossref Scopus (121) Google Scholar). The penicillanic sulfones do not require protonation to catalyze the β-elimination event and opening of the five-membered ring (8Imtiaz U. Billings E.M. Knox J.R. Mobashery S. Biochemistry. 1994; 33: 5728-5738Crossref PubMed Scopus (76) Google Scholar). This might account for the activity of tazobactam against the CA-resistant class A β-lactamases. Our studies were designed to provide detailed evidence regarding the mechanism of inhibition of class A β-lactamases by tazobactam, a penicillanic sulfone inhibitor. ESI/MS, HPLC/ESI/MS, and HPLC/ESI/MS/MS techniques were employed to identify the amino acid residues of PC1 directly involved in inactivation by tazobactam. In addition, the reaction products of tazobactam, following the initial acylation of the enzyme, were examined. From these findings, a mechanistic scheme for the inactivation of class A β-lactamases by TZB was proposed. The similarities and differences in the inactivation of β-lactamases by clavulanic acid and the penicillanic acid sulfones are discussed. PC1 β-lactamase (256 residues, MW 28,794.3 (10Ambler R.P. Biochem. J. 1975; 151: 197-218Crossref PubMed Scopus (58) Google Scholar,11McLaughlin J.R. Murray C.J. Rabinowitz J.C. J. Biol. Chem. 1981; 256: 11272-11282PubMed Google Scholar)) was produced by Proton Products (Berkshire, UK). TEM-1 β-lactamase (263 residues, MW 28,909.8 (12Sutcliffe J.G. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3731-3741Crossref Scopus (599) Google Scholar)) was extracted from a recombinant high level overexpressing Escherichia coli strain (13Rasmussen B.A. Gluzman Y. Tally F.P. Mol. Microbiol. 1991; 5: 1211-1219Crossref PubMed Scopus (29) Google Scholar) and was purified to near homogeneity using Sephadex G-75 column chromatography. Sequencing grade endoproteinases, Glu-C and trypsin (modified), were purchased from Roche Molecular Biochemicals. Ambler's numbering system for the amino acid sequence of PC1 (14Ambler R.P. Coulson A.F.W. Frere J.-M. Ghuysen J.-M. Joris B. Forsman M. Levesque R.C. Tiraby G. Waley S.G. Biochemistry J. 1991; 276: 269-272Crossref PubMed Scopus (854) Google Scholar) was used throughout the text. The free acid form of TZB (MW 300) was synthesized at Wyeth-Ayerst Research (Pearl River, NY). TZB was prepared as a 2 mm stock solution in HPLC grade water unless otherwise indicated. For intact protein analysis, enzyme stock solutions (100 μm) of PC1 and TEM-1 were prepared in HPLC grade water. Samples of TZB reacted with PC1 and TEM-1, respectively, were prepared by incubating the enzyme with inhibitor at inhibitor/enzyme molar ratios of 10:1 and 100:1, respectively, in H2O for 60 min at 25 °C. Greater than 90% inhibition of PC1 and TEM-1 by TZB, respectively, was confirmed by testing for enzyme activity using the chromogenic substrate nitrocefin and monitoring at 495 nm using a Beckman Model DU7400 spectrophotometer (15O'Callanghan C.H. Morris A. Kirby S. Single A.H. Antimicrob. Agents Chemother. 1972; 1: 283-288Crossref PubMed Scopus (1477) Google Scholar). To verify the specificity of the reaction of TZB with PC1 and TEM-1, the enzymes were denatured using 2% formic acid in H2O: acetonitrile (H2O:ACN, 1:1 v/v) for 10 min prior to incubation with TZB. The endoproteinase Glu-C was used to cleave the peptide bonds C-terminally at glutamic acid residues. PC1 and TZB were dissolved in 20 mm ammonium carbonate, pH 7.8. TZB and PC1 were mixed (inhibitor:enzyme, 10:1, m/m) and incubated for 60 min at 25 °C. PC1 hydrolytic activity was inhibited to greater than 90% by TZB. This was confirmed by testing for residual enzyme activity using nitrocefin (as above). The TZB-PC1 reaction mixture and PC1 control (without TZB) were each individually digested with Glu-C at a molar ratio of 40:1 (PC1:Glu-C) for 4 h at 25 °C. Digestion with trypsin was performed using sequencing grade, modified trypsin protease. PC1 and TZB were dissolved in 100 mm Tris/HCl, pH 8.5, at concentrations of 50 and 2000 μm, respectively. TZB and PC1 were mixed (inhibitor:enzyme, 10:1, m/m) and incubated for 60 min at 25 °C. Inactivation of enzymatic activity by TZB was confirmed (as above). The TZB-PC1 reaction mixture and PC1 control (without TZB) were each individually digested with trypsin at a molar ratio of 40:1 (PC1:trypsin) for 10 h at 37 °C. Electrospray ionization mass spectra were obtained in the positive and negative ion modes with a Micromass Quattro triple quadrupole mass spectrometer and a Platform II single quadrupole mass spectrometer. The samples were prepared at ∼10 pmol/μl in 3% acetic acid in H2O:ACN (1:1, v/v). Samples were flow injected (2–5 μl) into the source of the mass spectrometer at a rate of 10 μl/min utilizing a carrier solvent of H2O:ACN (1:1, v/v). Protein data were acquired over a wide scan mass range of m/z ∼500–1600 with 16 points/m/z or over a narrow scan mass range ofm/z 800–1000 at 64 data points/m/z, with a scan duration of 15–25 s. The cone voltage was set to 50 V. Nitrogen was used as the nebulizing and drying gas with flow rates of 0.2 and 5 liters/min, respectively. Ten to twenty spectra were averaged, smoothed, baseline subtracted, and using the Micromass The mass spectrometer was with HPLC/ESI/MS/MS spectra were obtained with the Micromass Quattro triple quadrupole mass spectrometer using a of 50 with as the gas at mass spectra were obtained in the negative ion using a high performance mass spectrometer with a fast atom The used was a mixture of and data were acquired by at mass data were acquired by at a of The negative ion the following for wide mass range and and for narrow mass range A Model HPLC and set to spectra over the nm were employed in all A solvent of from to containing acid was over 60 min at a mm An ratio of was to 50 μl/min to the ion source of a Micromass mass spectrometer. Nitrogen was used as the nebulizing and drying spectra were acquired over a range of m/z at using electrospray ionization in the positive ion with a cone voltage of V. Ten of Glu-C or PC1 and TZB-PC1 were HPLC/ESI/MS/MS was performed the and with the mass spectrometer as in the spectra were acquired using an over a range of m/z 50 to and was used as the The used for analysis of the data followed the of and J. PubMed Scopus Google Scholar). For protein β-lactamases and TZB were dissolved in HPLC grade water. the pH of the reaction mixture was PC1 and TEM-1 enzymes in water as as in a control mm pH the incubation water of a system provided mass ESI/MS The acid form of TZB was for reactions ESI/MS to the formation of were observed using the form of TZB. The mass spectra were obtained in the positive ion narrow scan high and the data were with a the mass spectra for PC1 MW and the TZB-PC1 reaction products For in to the PC1 MW new components were observed to the mass of PC1 mass of and Da and 2 the mass spectra of TEM-1 2 MW and reaction products 2 For in to TEM-1 MW three new components were observed to the mass of TEM-1 mass of and Da and the observed masses and for the products following the reaction of TZB with PC1 and TEM-1, respectively. The of the new products in and 2 the of formation of the reaction intermediates the ESI/MS spectra for TEM-1 and TEM-1 (MW reaction TEM-1 (MW and TZB-modified TEM-1 with mass of and masses for the reaction of PC1 and TEM-1 with TZB and proposed to enzyme mass structure mass 28,794.3 10:1 2 mass 28,909.8 mass 28,794.3 (10Ambler R.P. Biochem. J. 1975; 151: 197-218Crossref PubMed Scopus (58) Google Scholar,11McLaughlin J.R. Murray C.J. Rabinowitz J.C. J. Biol. Chem. 1981; 256: 11272-11282PubMed Google mass 28,909.8 (12Sutcliffe J.G. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3731-3741Crossref Scopus (599) Google Scholar). in a new Following PC1 inactivation by TZB, three mass components were observed in the positive and negative ion electrospray three components have of 248, 264, and respectively. masses were observed by mass spectrometry in the negative ion mass of the observed for the three components were m/z and with and respectively. with electrospray data in the positive and negative ion modes the of three proposed to TZB reaction products and see The formation of these was of the pH used denatured PC1 and TEM-1 by to 2% formic were each reacted with TZB, new mass components were observed by the masses to the of PC1 and TEM-1 were that the active are for the specificity of the the UV for the data for PC1 and TZB-PC1 The of the are in data for the PC1 at at m/z The data a molecular weight of the molecular weight of for a peptide composed of amino acid residues containing Ser-130. The data for the TZB-PC1 in to 2 with MW a as with at an ion with a molecular weight of This MW to a mass of 70 Da to the mass of the PC1 peptide containing amino acid residues for analysis of PC1 and TZB-PC1 digested with endoproteinase acid 2 studied by studied by 70 to and were to et al. 58–82 acid to were to et 58–82 58–82 70 58–82 88 58–82 studied by to and were to et al. J.R. Murray C.J. Rabinowitz J.C. J. Biol. Chem. 1981; 256: 11272-11282PubMed Google acid to were to et J.R. Murray C.J. Rabinowitz J.C. J. Biol. Chem. 1981; 256: 11272-11282PubMed Google Scholar). in a new data for PC1 that and at and at The indicated the molecular weight of this peptide to This observed MW the molecular weight of for a peptide containing amino acid residues 58–82 The data for the TZB-PC1 three components at min The of produced by these components of and 4 and to 52, 70, and 88 Da mass to the mass of the PC1 peptide composed of amino acid residues at at m/z and In the has a molecular weight of to the Da peptide composed of amino acid residues The analysis of TZB-PC1 that TZB fragments were bound to peptides composed of residues containing Ser-70, and residues containing Ser-130. data for PC1 and TZB-PC1 are in data for trypsin digested PC1 and TZB-PC1 that at of an ion at m/z to a peptide composed of amino acid residues data for trypsin digested TZB-PC1 that at of an ion at m/z The of 70 Da the masses of and indicated that a 70-Da fragment of TZB was bound to one of the amino acid residues was not observed in the data for for analysis of PC1 and TZB-PC1 digested with acid studied by studied by 70 or studied by in a new The peptides to 2 II and and II and from PC1 and respectively, following with were using The of these two peptides were confirmed to that of peptides composed of amino acid residues 125–141. a the of the ion at m/z to 2 II and from PC1. The masses of the from were with that of a peptide composed of residues in 5 The of the ion at m/z to II and of the modified peptide from is in 5 This has a to that of the peptide composed of amino acid residues 125–141. The of from were observed in An fragment ion at m/z was observed for the that Ser-130 was the of the 70-Da TZB The peptides to and from PC1 and respectively, following with were by The of these peptides were confirmed to amino acid residues The modified by TZB was the spectra for and The masses of the from in spectra are with the amino acid sequence of residues 2 in The TZB moiety of the modified peptide is in the in a at m/z a 70-Da mass component than the ion The TZB-PC1 adduct is The the loss of the TZB moiety prior to the the of the 70 are and peptides were Chem. Scopus Google Scholar). Two fragment 70 and 70 were observed in the for the TZB-modified peptide that Ser-70 is the site of by the TZB a of fragment were observed with in to fragment ion was observed at This represented an mass loss from the peptide composed of residues at m/z This is in with the of a peptide composed of residues 58–82 in the complex and 4 In addition, fragment with an mass loss for the were observed at m/z with at m/z with at m/z with and at m/z with The mass loss fragments were observed with the mass fragment containing Ser-70 not with the mass fragment Ser-70, and The of these in the peptide and the in the that is to the of the 70-Da fragment from TZB. is that the HPLC/ESI/MS/MS the TZB adduct is from the peptide as an fragment that an from the attached this is for the of the TZB adduct to ESI/MS has to a in the molecular of This ionization has been to the of covalent β-lactamase substrate and (6Brown R.P. A Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar, R. C. C. and S. G. J. Chem. Soc. Chem. Scholar, O. J.-M. PubMed Scopus Google Scholar, D.J. B. R. Y. J. Antimicrob. Agents Chemother. 1997; PubMed Google Scholar). an analysis of the inactivation mechanism of the potent penicillanic sulfone β-lactamase tazobactam, on the class A β-lactamases, PC1 and TEM-1, using of mass ESI/MS was used to identify the masses of the TZB fragments following reaction with the confirmed the of ESI/MS and the TZB-modified PC1 peptides composed of amino acid residues and 125–141. In of HPLC and amino acid sequence analysis, HPLC/ESI/MS/MS was used to provide evidence for the PC1 residues, Ser-70 and Ser-130. The data are the of the mechanism of inactivation by a penicillanic sulfone inhibitor with class A β-lactamases and are the on mass spectrometry A scheme the of inactivation of class A β-lactamases by TZB is proposed and is to with the mass spectrometry data M. B. and M. in of the on Agents and for Scholar). The increased masses observed by ESI/MS of and Da for TZB-PC1 were by performed on the digested TZB-PC1 The to the formation of proposed propiolylated enzyme that have formed from decomposition of intermediates and The mass of Da to intermediates HPLC/ESI/MS/MS analysis indicated that Da at Ser-70, and The to the ESI/MS and components with molecular masses of 248, 264, and 280 Da were as products of the reaction of TZB with PC1. masses for these reaction products the of molecular and proposed and K. Yang Y. Rasmussen B.A. Shlaes of the for and for Scholar). The observed ESI/MS, and data were with the of a acid (MW to form the products of (MW and (MW K. Yang Y. Rasmussen B.A. Shlaes of the for and for Scholar). are with the rapid of the TZB adduct 2 to intermediates followed by the loss of from or enamines 4 and the components observed in the protein by ESI/MS analysis of TZB-PC1 was a component with a mass of Da to PC1. This mass component to some or all of the proposed reaction intermediates This is with the proposed mechanistic scheme TZB and the enzyme form the acyl-enzyme is to that formed with the β-lactam of these This adduct was not observed in ESI/MS analysis of the reaction the rapid loss of and formation of products and the TZB-PC1 β-elimination in the opening of the sulfone containing the five-membered ring to form the imine acyl-enzyme complex to the and UV studies of TZB-PC1 (7Bush K. Macalintal C. Rasmussen B.A. Lee V. Yang Y. Antimicrob. Agents Chemother. 1993; 37: 851-858Crossref PubMed Scopus (235) Google Scholar) are with the formation of imine and enamine of the β-lactamase inhibitor. The observed TZB-PC1 adduct with a mass the propiolylated enzyme, to from 4 In addition, of Following the initial of Ser-130 with intermediates 5 and the displacement of form a cross-linked vinyl of structure of the imine and enamine enzyme and of to of hydrolysis of of the inhibitor and active of the observed reaction products of TEM-2 (6Brown R.P. A Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar) were in TZB inactivation products of class A β-lactamases, the imine and enamine intermediates. The residues, Ser-70 and proposed by HPLC/ESI/MS/MS data for are to the residues in TEM-2 for In the the component that a mass was in the This is likely the adduct represented by structure However, were following and In this a adduct was for the TZB-PC1 peptide composed of amino acid residues This component is most likely represented by structure in The of the vinyl cross-linked with Ser-70 and as an in β-lactamase inactivation by TZB, was not by followed by The of Ser-130 in the inactivation of class A β-lactamases by CA was by et al. (9Imtiaz U. Billings E.M. Knox J.R. Manavathu E., K. Lerner S.A. Mobashery S. J. Am. Chem. Soc. 1993; 115: 4435-4442Crossref Scopus (121) Google Scholar). It was proposed that Ser-130 the of the by to the β-lactam In of al. (6Brown R.P. A Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar) a 70-Da adduct attached to the TEM-2 peptide composed of amino acid residues The 70-Da TZB fragment, attached to the peptide composed of PC1 amino acid residues was Glu-C of of the fragment to Ser-130 was by the analysis of the HPLC/ESI/MS/MS Ser-130 is directly involved in β-lactamase inactivation by sulfone In that clavams and sulfones are structurally the inactivation of class A β-lactamases by CA and TZB are the formation of the