- Email: [email protected]
A rat monoclonal antibody that catalyses the hydrolysis of a nitrophenyl-β-lactam
BBRC Biochemical and Biophysical Research Communications 299 (2002) 273–276 www.academicpress.com
A rat monoclonal antibody that catalyses the hydrolysis of a nitrophenyl-b-lactam Elizabeth L. Ostler,a Christopher J. Dean,b Nicola Barber,a Maurizio Valeri,b Stuart James,a Marina Resmini,c Guillaume Boucher,a Nickolas Romanov,d Keith Brocklehurst,e and Gerard Gallachera,* a
School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Moulsecoomb, Brighton BN2 4GJ, UK b Institute of Cancer Research, Hybridoma Unit, McElwain Laboratory, 15, Cotswold Road, Sutton, Surrey, UK c Department of Chemistry, Queen Mary, University of London, Mile End Rd, London E1 4NS, UK d Erevanskkaya 14-a, K25, 03087, Kiev 87, Ukraine e Laboratory of Structural and Mechanistic Enzymology, School of Biological Sciences, Queen Mary, University of London, Mile End Rd, London E1 4NS, UK Received 10 October 2002
Abstract We report the first example of a monoclonal antibody-catalysed hydrolysis of a b-lactam where the antibodies were generated by a simple transition-state analogue. A rat monoclonal antibody (1/91c/4d/26) generated by using an acyclic 4-nitrophenylphosphate immunogen catalysed the hydrolysis of corresponding 4-nitrophenyl carbonates but, more importantly, also catalysed the hydrolysis of N-(4-nitrophenyl)-azetidinone at pH 8 with kcat ¼ 8:7 106 s1 and KM ¼ 35 lM. This is the first example of a rat monoclonal catalytic antibody. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Catalytic antibodies; b-Lactam; Hydrolysis; Monoclonal antibodies; Abzyme
In the 16 years since catalytic antibodies were first reported [1,2] there has been rapid growth with large numbers of new catalytic antibodies being described [3,4], the majority of which are esterolytic. Amide hydrolysis is a more energetically demanding reaction and only a very small number of antibodies that catalyse the hydrolysis of amides have been reported [5–11]. The production of catalytic antibodies for amide hydrolysis is consequently the focus of considerable current effort [12] and success promises a wealth of applications in medicine and biotechnology. Some time ago we reported that polyclonal antibodies generated by the phosphate immunogen 1a catalyse the hydrolyses of the carbonates 2a and 2b [13,14] and more recently we revealed that they also catalyse the hydrolysis of the structurally
* Corresponding author. Fax: +44-1273-679-333. E-mail address: [email protected] (G. Gallacher).
related b-lactam 3 [15]. We have now augmented this recent work by producing a monoclonal antibody that catalyses the hydrolyses of these same substrates including the important ‘‘amide’’ bond-containing b-lactam 3. This result is important in demonstrating the similarity of polyclonal and monoclonal catalytic antibodies generated by the same hapten, and is the first example of the use of a simple transition-state analogue to generate a monoclonal antibody that catalyses the hydrolysis of a b-lactam. It is also the first example of a rat monoclonal catalytic antibody.
Materials and methods Materials. Solvents (analytical grade), aluminium-backed silica gel 60 TLC plates (Merck 5554), and silica gel 60 for flash chromatography (Merck particle size 0.040–0.063 mm) were from Merck (Lutterworth, Leics., UK). All other chemicals were from Sigma–Aldrich (Poole, Dorset, UK).
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 6 0 4 - 9
274
E.L. Ostler et al. / Biochemical and Biophysical Research Communications 299 (2002) 273–276
Results
Syntheses. The phosphate immunogen 1a, transition-state analogue 1c and carbonates (2a, 2b, and 4) were synthesised as described previously [14]. 4-Nitrophenyl azetidinone 3 was synthesised according to literature procedures [16]. Production and purification of antibodies. The phosphate immunogen 1a was used to generate monoclonal antibodies in three rats, by following established methods [17]. Hybridomas which secreted antibodies capable of binding the hapten 1a were identified by using an immunoradiometric assay (IRMA) and binding titres in the assay were used to select one clone for further investigation. This clone was cultured in a rolling culture bottle and the monoclonal antibody (1/91c/ 4d/26) was isolated by salt fractionation. Isotyping experiments showed that this is an IgG antibody of subclass 1, and it was purified via immunoaffinity purification on a mouse anti-rat-c-1 affinity column. Kinetics of hydrolysis. Steady-state kinetics of the hydrolysis of carbonate 2b were carried out as described previously [14]. Initial rates of the hydrolysis the b-lactam substrate were determined by using a Perkin–Elmer Lambda 2 dual-beam spectrophotometer to monitor the linear change in UV absorbance (versus matched reference solutions containing no b-lactam) at both 327 and 410 nm. UV Absorbance was recorded at intervals of 3 min over 8 h at a constant temperature of either 33 or 37 °C. These values were corrected for aqueous (non-catalysed) hydrolysis determined in the absence of the antibody, under otherwise identical experimental conditions. Observed initial rates of the non-catalysed reaction have been shown previously [15] to be indistinguishable from those determined in the presence of IgG from non-immunised animals. Values of Vmax and KM were then determined by using the unweighted non-linear regression program of GraphPad Prism v 3.0. Values of the catalytic rate constant (kcat ) were calculated by using kcat ¼ Vmax /(2[IgG]).
Hydrolysis of the carbonate 2a is accelerated by the monoclonal antibody (1/91c/4d/26) at pH 8, and this rate acceleration is completely inhibited by the phosphate transition-state analogue 1b, demonstrating that the observed activity is due to the antibody paratope. Hydrolysis of the isomeric 2-nitrophenyl carbonate 4 is not catalysed by the antibody. This provides additional strong evidence that the observed catalysis is due to the antibody and is not a result of any possible contaminants [13]. The carbonate substrate 2a is rather hydrophobic, and its limited solubility in water hindered an accurate determination of KM for the catalysed reaction. In previous polyclonal catalytic antibody investigations we described [14] the design and synthesis of the closely related but more soluble carbonate substrate 2b. Hydrolysis of the more soluble carbonate 2b was also catalysed by the monoclonal antibody (1/91c/ 4d/26) with Michaelian catalytic properties. A plot of initial rate for the hydrolysis of the carbonate 2b (corrected for non-catalysed hydrolysis) against substrate concentration, at pH 8 and 25 °C, is shown in Fig. 1. Analysis of the saturation curve gives KM ¼ 34 lM and kcat ¼ 0:3 s1 (taking the concentration of catalytic sites ¼ 2 ½IgG). The non-catalysed hydrolysis has an apparent first-order kinetic constant knon-cat ¼ 2 104 s1 at pH 8 and 25 °C. The rate constant ratio kcat =knon-cat is frequently [4] used for comparison of the activities of different catalytic antibodies. The (1/91c/4d/ 26) monoclonal antibody-catalysed hydrolysis of the carbonate 2b has kcat =knon-cat ¼ 1:5 103 . This is similar
Fig. 1. Plot of initial rate against concentration of carbonate 2b. The points represent values corrected for uncatalysed hydrolysis at 25 °C, in pH 8 sodium phosphate buffer (0.05 M) containing 0.67% v/v acetonitrile, in the presence of 0.2 lM IgG. The curve represents best fit values of Vmax and KM obtained by fitting the data to the Michaelis– Menten equation using an unweighted least-squares analysis (r2 ¼ 0:97).
E.L. Ostler et al. / Biochemical and Biophysical Research Communications 299 (2002) 273–276
to the estimated [18] intrinsic activity of our polyclonal catalytic antibodies, and is typical of a very good, though not extraordinary, catalytic antibody. A comparison may be made with earlier [19] mouse monoclonal catalytic antibodies generated in vitro by the hapten 1c. These antibodies catalysed the hydrolysis of the carbonate 2a with kcat =knon-cat values between 2:5 103 and 5:5 103 . Another interesting comparison is with the polyclonal catalytic antibody preparation, 271-22 [20] generated by the immunogen 1a. This preparation catalyses the hydrolysis of the carbonate 2a with kcat =knon-cat ¼ 1:15 102 for the total IgG. Kinetic analysis has more recently [18] established that this polyclonal preparation contains at most 8% and probably less than 1% catalytically active IgG. This means that the antibody catalysts within the IgG have a kcat =knon-cat value of at least 1:45 103 and probably greater than 1:2 104 . The same argument applied to the most active preparation PCA 270-29 [21] gives an intrinsic kcat =knon-cat value greater than 6:8 104 . A more significant finding was that the monoclonal antibodies (1/91c/4d/26) also catalyse the hydrolysis of the b-lactam 3. This ‘‘amide’’ substrate has the following advantages for catalytic antibody investigations: (i) it is a reactive amide whose hydrolysis is activated by both strain and electronic effects, (ii) the product of its hydrolysis, the amino acid carboxylate 5, contains a nitroaniline chromophore which facilitates kinetic measurements, (iii) its base promoted hydrolysis, in the absence of catalyst, is well characterised [22–24], (iv) the nitrophenyl group provides recognition and binding in antibody studies, (v) product inhibition is unlikely because the ring opened product is conformationally very different from substrate and phosphate transition-state analogues, and (vi) it is intermediate in reactivity between a nitrophenyl ester and a nitroanilide. The blactam 3 has a knon-cat ¼ 1 107 s1 at pH 8 and 25 °C, which is some 2000 times lower than that of the carbonate 2b. A plot of initial rate for the catalysed hydrolysis of the b-lactam 3 (corrected for non-catalysed hydrolysis) against substrate concentration, at pH 8, and 33 °C, is shown in Fig. 2. Analysis of the saturation curve gives kcat ¼ 8:7 106 s1 and Km ¼ 35 lM (taking the concentration of catalytic sites ¼ 2 ½IgG). An equivalent set of experiments carried out at pH 9 and 37 °C also gave a saturation curve, analysis of which gives kcat ¼ 9:5 105 s1 and kM ¼ 56 lM. The non-catalysed hydrolysis has apparent first-order kinetic constants, knon-cat ¼ 3:5 107 s1 at pH 8 and 33 °C, and 4:6 106 s1 at pH 9 and 37 °C. Hence kcat =knon-cat ratios for the above experiments are 25 and 20, respectively. The catalysed reaction was completely inhibited by the hapten (1b), again confirming that the catalysis is antibody-mediated.
275
Fig. 2. Plot of initial rate against concentration of b-lactam 3. The points represent values obtained in initial rate experiments (corrected for uncatalysed hydrolysis) in pH 8 sodium phosphate buffer (0.05 M) containing 0.67% v/v acetonitrile, at 33 °C, in the presence of 0.86 lM IgG. The curve represents best fit values of Vmax and KM obtained by fitting the data to the Michaelis–Menten equation using an unweighted least-squares analysis (r2 ¼ 0:98).
Discussion This is the first report of rat monoclonal catalytic antibodies and the first report of monoclonal b-lactamase antibodies generated by immunisation with a transition-state analogue. For the important b-lactam substrate 3, a direct comparison may be made between the values obtained for the monoclonal catalytic antibodies at pH 9 and 37 °C and the published [15] values for the polyclonal catalytic antibodies under identical conditions. The monoclonal catalytic antibodies give a measured catalytic rate constant kcat ¼ 9:5 105 s1 , which is remarkably similar to the estimated catalytic rate constant kcat ¼ 1:6 104 s1 [15] for the catalytic fraction of the earlier sheep polyclonal preparation. However, since the estimate was based on the upper limit of the proportion of catalytic antibodies within the total IgG, it is probable that the polyclonal IgG contains catalytic antibodies of considerably greater activity. Neither the polyclonal nor the monoclonal antibodies were expected to catalyse the hydrolysis of the b-lactam, since the phosphate immunogen 1a was designed to generate antibodies that would catalyse the hydrolysis of carbonates such as 2a and 2b. The b-lactam 3 lacks major parts of the structural features expected to contribute to recognition and binding. In addition, the blactam ring is expected to present steric hindrance to binding and has N–C–O bond angles (in the ground state and transition state) that deviate from the equivalent O–C–O bond angles (in the carbonate ground state and transition state) and the O–P–O angle in the hapten. It is likely, therefore, considering the quantitative and qualitative similarity of the catalytic activities observed, that similar modes of binding may be operating in the
276
E.L. Ostler et al. / Biochemical and Biophysical Research Communications 299 (2002) 273–276
rat monoclonal antibody and the catalytic fraction of the sheep polyclonal antibody preparation. Only seven monoclonal antibodies (or antibody fragments) have been generated that catalyse the hydrolysis of amides. Of these, only two catalyse the hydrolysis of b-lactams and neither was generated by immunisation with a transition-state analogue. The results described above add one more monoclonal catalytic antibody and this was generated by using a transition-state analogue. We predict that analogues that are more closely related to the transition-state of blactam hydrolysis (for example, cyclic phosphonates or phosphonamidates) will generate monoclonal antibodies with greater b-lactamase activity. The earlier polyclonal antibody studies were valuable in uncovering b-lactamase activity where it was not expected. The monoclonal studies, described above, have confirmed the polyclonal results and demonstrated the similar properties of antibodies from different sources. Monoclonal antibodies are more amenable to structure determination and detailed investigations of the modes of b-lactam binding than are polyclonal antibodies. This avenue of investigation will be the subject of future experiments.
Acknowledgments We thank The Wellcome Trust for financial support (Grant reference 060295). Guillaume Boucher was supported by a TMR award (ERBFMRXCT 980 193) from the European Commission.
References [1] A. Tramontano, K.D. Janda, R.A. Lerner, Catalytic antibodies, Science 234 (1986) 1566–1570. [2] S.J. Pollack, J.W. Jacobs, P.G. Schultz, Selective chemical catalysis by an antibody, Science 234 (1986) 1570–1573. [3] R.A. Lerner, S.J. Benkovic, P.G. Schultz, At the crossroads of chemistry and immunology—catalytic antibodies, Science 252 (1991) 659–667. [4] J.D. Stevenson, N.R. Thomas, Catalytic antibodies and other biomimetic catalysts, Nat. Prod. Rep. 17 (2000) 535–577. [5] K.D. Janda, D. Schloeder, S.J. Benkovic, R.A. Lerner, Induction of an antibody that catalyzes the hydrolysis of an amide bond, Science 241 (1988) 1188–1191. [6] M.T. Martin, T.S. Angeles, R. Sugasawara, N.I. Aman, A.D. Napper, M.J. Darsley, R.I. Sanchez, P. Booth, R.C. Titmas, Antibody-catalyzed hydrolysis of an unsubstituted amide, J. Am. Chem. Soc. 116 (1994) 6508–6512.
[7] F. Benedetti, F. Berti, A. Colombatti, C. Ebert, P. Linda, F. Tonizzo, Anti-sulfonamide antibodies catalyse the hydrolysis of a heterocyclic amide, Chem. Commun. (1996) 1417–1418. [8] C.S. Gao, B.J. Lavey, C.H.L. Lo, A. Datta, P. Wentworth, K.D. Janda, Direct selection for catalysis from combinatorial antibody libraries using a boronic acid probe: primary amide bond hydrolysis, J. Am. Chem. Soc. 120 (1998) 2211–2217. [9] B. Gong, P.G. Schultz, S.S. Yoon, A catalytic antibody for the hydrolysis of peptides, J. Korean Chem. Soc. 42 (1998) 462–466. [10] B. Avalle, D. Thomas, A. Friboulet, Functional mimicry: elicitation of a monoclonal anti-idiotypic antibody hydrolyzing b-lactams, FASEB J. 12 (1998) 1055–1060. [11] F. Tanaka, H. Almer, R.A. Lerner, C.F. Barbas, Catalytic singlechain antibodies possessing b-lactamase activity selected from a phage displayed combinatorial library using a mechanism-based inhibitor, Tetrahedron Lett. 40 (1999) 8063–8066. [12] G.M. Blackburn, S.X. Deng, Catalytic antibodies for the hydrolysis of unactivated peptides, Biochem. Soc. Trans. 21 (1993) 1102–1107. [13] G. Gallacher, C.S. Jackson, M. Searcey, G.T. Badman, R. Goel, C.M. Topham, G.W. Mellor, K. Brocklehurst, A polyclonal antibody preparation with Michaelian catalytic properties, Biochem. J. 279 (1991) 871–881. [14] M. Resmini, R. Vigna, C. Simms, N.J. Barber, E.P. HagiPavli, A.B. Watts, C. Verma, G. Gallacher, K. Brocklehurst, Characterisation of the hydrolytic activity of a polyclonal catalytic antibody preparation by pH-dependence and chemical modification studies: evidence for the involvement of Tyr and Arg side chains as hydrogen-bond donors, Biochem. J. 326 (1997) 279–287. [15] E.L. Ostler, M. Resmini, G. Boucher, N. Romanov, K. Brocklehurst, G. Gallacher, Polyclonal antibody-catalysed hydrolysis of a b-lactam, Chem. Commun. (2002) 226–227. [16] H. Takahata, Y. Ohnishi, T. Yamazaki, A convenient synthesis of monocyclic b-lactams, Heterocycles 14 (4) (1980) 467–469. [17] C.J. Dean, Methods Mol. Biol. 10 (1992) 43–63. [18] M. Resmini, S. Gul, S. Carter, S. Sonkaria, C.M. Topham, G. Gallacher, K. Brocklehurst, A general kinetic approach to investigation of active-site availability in macromolecular catalysts, Biochem. J. 346 (2000) 117–125. [19] M. Stahl, B. Goldie, S.P. Bourne, N.R. Thomas, Catalytic antibodies generated by in-vitro immunization, J. Am. Chem. Soc. 117 (1995) 5164–5165. [20] G. Gallacher, C.S. Jackson, M. Searcey, R. Goel, G.W. Mellor, C.Z. Smith, K. Brocklehurst, Catalytic antibody-activity elicited by active immunization—evidence for natural variation involving preferential stabilization of the transition-state, Eur. J. Biochem. 214 (1993) 197–207. [21] G. Gallacher, M. Searcey, C.S. Jackson, K. Brocklehurst, Polyclonal antibody-catalyzed amide hydrolysis, Biochem. J. 284 (1992) 675–680. [22] G.M. Blackburn, J.D. Plackett, Strain effects in acyl transfer reactions. Part 1. The kinetics of hydrolysis of some N-aryl lactams, J. Chem. Soc., Perkin Trans. 2 (1973) 1366–1371. [23] T.J. Broxton, L.W. Deady, Mechanism of the basic methanolysis of benzanilides, J. Org. Chem. 39 (1974) 2767. [24] A.R. Butler, K.A. Freeman, D.E. Wright, A study of factors affecting the rates of hydrolysis of some N-aryl-azetidin-2-ones (Naryl-b-lactams), J. Chem. Soc., Perkin Trans. 2 (1977) 765–769.
A rat monoclonal antibody that catalyses the hydrolysis of a nitrophenyl-b-lactam Elizabeth L. Ostler,a Christopher J. Dean,b Nicola Barber,a Maurizio Valeri,b Stuart James,a Marina Resmini,c Guillaume Boucher,a Nickolas Romanov,d Keith Brocklehurst,e and Gerard Gallachera,* a
School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Moulsecoomb, Brighton BN2 4GJ, UK b Institute of Cancer Research, Hybridoma Unit, McElwain Laboratory, 15, Cotswold Road, Sutton, Surrey, UK c Department of Chemistry, Queen Mary, University of London, Mile End Rd, London E1 4NS, UK d Erevanskkaya 14-a, K25, 03087, Kiev 87, Ukraine e Laboratory of Structural and Mechanistic Enzymology, School of Biological Sciences, Queen Mary, University of London, Mile End Rd, London E1 4NS, UK Received 10 October 2002
Abstract We report the first example of a monoclonal antibody-catalysed hydrolysis of a b-lactam where the antibodies were generated by a simple transition-state analogue. A rat monoclonal antibody (1/91c/4d/26) generated by using an acyclic 4-nitrophenylphosphate immunogen catalysed the hydrolysis of corresponding 4-nitrophenyl carbonates but, more importantly, also catalysed the hydrolysis of N-(4-nitrophenyl)-azetidinone at pH 8 with kcat ¼ 8:7 106 s1 and KM ¼ 35 lM. This is the first example of a rat monoclonal catalytic antibody. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Catalytic antibodies; b-Lactam; Hydrolysis; Monoclonal antibodies; Abzyme
In the 16 years since catalytic antibodies were first reported [1,2] there has been rapid growth with large numbers of new catalytic antibodies being described [3,4], the majority of which are esterolytic. Amide hydrolysis is a more energetically demanding reaction and only a very small number of antibodies that catalyse the hydrolysis of amides have been reported [5–11]. The production of catalytic antibodies for amide hydrolysis is consequently the focus of considerable current effort [12] and success promises a wealth of applications in medicine and biotechnology. Some time ago we reported that polyclonal antibodies generated by the phosphate immunogen 1a catalyse the hydrolyses of the carbonates 2a and 2b [13,14] and more recently we revealed that they also catalyse the hydrolysis of the structurally
* Corresponding author. Fax: +44-1273-679-333. E-mail address: [email protected] (G. Gallacher).
related b-lactam 3 [15]. We have now augmented this recent work by producing a monoclonal antibody that catalyses the hydrolyses of these same substrates including the important ‘‘amide’’ bond-containing b-lactam 3. This result is important in demonstrating the similarity of polyclonal and monoclonal catalytic antibodies generated by the same hapten, and is the first example of the use of a simple transition-state analogue to generate a monoclonal antibody that catalyses the hydrolysis of a b-lactam. It is also the first example of a rat monoclonal catalytic antibody.
Materials and methods Materials. Solvents (analytical grade), aluminium-backed silica gel 60 TLC plates (Merck 5554), and silica gel 60 for flash chromatography (Merck particle size 0.040–0.063 mm) were from Merck (Lutterworth, Leics., UK). All other chemicals were from Sigma–Aldrich (Poole, Dorset, UK).
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 6 0 4 - 9
274
E.L. Ostler et al. / Biochemical and Biophysical Research Communications 299 (2002) 273–276
Results
Syntheses. The phosphate immunogen 1a, transition-state analogue 1c and carbonates (2a, 2b, and 4) were synthesised as described previously [14]. 4-Nitrophenyl azetidinone 3 was synthesised according to literature procedures [16]. Production and purification of antibodies. The phosphate immunogen 1a was used to generate monoclonal antibodies in three rats, by following established methods [17]. Hybridomas which secreted antibodies capable of binding the hapten 1a were identified by using an immunoradiometric assay (IRMA) and binding titres in the assay were used to select one clone for further investigation. This clone was cultured in a rolling culture bottle and the monoclonal antibody (1/91c/ 4d/26) was isolated by salt fractionation. Isotyping experiments showed that this is an IgG antibody of subclass 1, and it was purified via immunoaffinity purification on a mouse anti-rat-c-1 affinity column. Kinetics of hydrolysis. Steady-state kinetics of the hydrolysis of carbonate 2b were carried out as described previously [14]. Initial rates of the hydrolysis the b-lactam substrate were determined by using a Perkin–Elmer Lambda 2 dual-beam spectrophotometer to monitor the linear change in UV absorbance (versus matched reference solutions containing no b-lactam) at both 327 and 410 nm. UV Absorbance was recorded at intervals of 3 min over 8 h at a constant temperature of either 33 or 37 °C. These values were corrected for aqueous (non-catalysed) hydrolysis determined in the absence of the antibody, under otherwise identical experimental conditions. Observed initial rates of the non-catalysed reaction have been shown previously [15] to be indistinguishable from those determined in the presence of IgG from non-immunised animals. Values of Vmax and KM were then determined by using the unweighted non-linear regression program of GraphPad Prism v 3.0. Values of the catalytic rate constant (kcat ) were calculated by using kcat ¼ Vmax /(2[IgG]).
Hydrolysis of the carbonate 2a is accelerated by the monoclonal antibody (1/91c/4d/26) at pH 8, and this rate acceleration is completely inhibited by the phosphate transition-state analogue 1b, demonstrating that the observed activity is due to the antibody paratope. Hydrolysis of the isomeric 2-nitrophenyl carbonate 4 is not catalysed by the antibody. This provides additional strong evidence that the observed catalysis is due to the antibody and is not a result of any possible contaminants [13]. The carbonate substrate 2a is rather hydrophobic, and its limited solubility in water hindered an accurate determination of KM for the catalysed reaction. In previous polyclonal catalytic antibody investigations we described [14] the design and synthesis of the closely related but more soluble carbonate substrate 2b. Hydrolysis of the more soluble carbonate 2b was also catalysed by the monoclonal antibody (1/91c/ 4d/26) with Michaelian catalytic properties. A plot of initial rate for the hydrolysis of the carbonate 2b (corrected for non-catalysed hydrolysis) against substrate concentration, at pH 8 and 25 °C, is shown in Fig. 1. Analysis of the saturation curve gives KM ¼ 34 lM and kcat ¼ 0:3 s1 (taking the concentration of catalytic sites ¼ 2 ½IgG). The non-catalysed hydrolysis has an apparent first-order kinetic constant knon-cat ¼ 2 104 s1 at pH 8 and 25 °C. The rate constant ratio kcat =knon-cat is frequently [4] used for comparison of the activities of different catalytic antibodies. The (1/91c/4d/ 26) monoclonal antibody-catalysed hydrolysis of the carbonate 2b has kcat =knon-cat ¼ 1:5 103 . This is similar
Fig. 1. Plot of initial rate against concentration of carbonate 2b. The points represent values corrected for uncatalysed hydrolysis at 25 °C, in pH 8 sodium phosphate buffer (0.05 M) containing 0.67% v/v acetonitrile, in the presence of 0.2 lM IgG. The curve represents best fit values of Vmax and KM obtained by fitting the data to the Michaelis– Menten equation using an unweighted least-squares analysis (r2 ¼ 0:97).
E.L. Ostler et al. / Biochemical and Biophysical Research Communications 299 (2002) 273–276
to the estimated [18] intrinsic activity of our polyclonal catalytic antibodies, and is typical of a very good, though not extraordinary, catalytic antibody. A comparison may be made with earlier [19] mouse monoclonal catalytic antibodies generated in vitro by the hapten 1c. These antibodies catalysed the hydrolysis of the carbonate 2a with kcat =knon-cat values between 2:5 103 and 5:5 103 . Another interesting comparison is with the polyclonal catalytic antibody preparation, 271-22 [20] generated by the immunogen 1a. This preparation catalyses the hydrolysis of the carbonate 2a with kcat =knon-cat ¼ 1:15 102 for the total IgG. Kinetic analysis has more recently [18] established that this polyclonal preparation contains at most 8% and probably less than 1% catalytically active IgG. This means that the antibody catalysts within the IgG have a kcat =knon-cat value of at least 1:45 103 and probably greater than 1:2 104 . The same argument applied to the most active preparation PCA 270-29 [21] gives an intrinsic kcat =knon-cat value greater than 6:8 104 . A more significant finding was that the monoclonal antibodies (1/91c/4d/26) also catalyse the hydrolysis of the b-lactam 3. This ‘‘amide’’ substrate has the following advantages for catalytic antibody investigations: (i) it is a reactive amide whose hydrolysis is activated by both strain and electronic effects, (ii) the product of its hydrolysis, the amino acid carboxylate 5, contains a nitroaniline chromophore which facilitates kinetic measurements, (iii) its base promoted hydrolysis, in the absence of catalyst, is well characterised [22–24], (iv) the nitrophenyl group provides recognition and binding in antibody studies, (v) product inhibition is unlikely because the ring opened product is conformationally very different from substrate and phosphate transition-state analogues, and (vi) it is intermediate in reactivity between a nitrophenyl ester and a nitroanilide. The blactam 3 has a knon-cat ¼ 1 107 s1 at pH 8 and 25 °C, which is some 2000 times lower than that of the carbonate 2b. A plot of initial rate for the catalysed hydrolysis of the b-lactam 3 (corrected for non-catalysed hydrolysis) against substrate concentration, at pH 8, and 33 °C, is shown in Fig. 2. Analysis of the saturation curve gives kcat ¼ 8:7 106 s1 and Km ¼ 35 lM (taking the concentration of catalytic sites ¼ 2 ½IgG). An equivalent set of experiments carried out at pH 9 and 37 °C also gave a saturation curve, analysis of which gives kcat ¼ 9:5 105 s1 and kM ¼ 56 lM. The non-catalysed hydrolysis has apparent first-order kinetic constants, knon-cat ¼ 3:5 107 s1 at pH 8 and 33 °C, and 4:6 106 s1 at pH 9 and 37 °C. Hence kcat =knon-cat ratios for the above experiments are 25 and 20, respectively. The catalysed reaction was completely inhibited by the hapten (1b), again confirming that the catalysis is antibody-mediated.
275
Fig. 2. Plot of initial rate against concentration of b-lactam 3. The points represent values obtained in initial rate experiments (corrected for uncatalysed hydrolysis) in pH 8 sodium phosphate buffer (0.05 M) containing 0.67% v/v acetonitrile, at 33 °C, in the presence of 0.86 lM IgG. The curve represents best fit values of Vmax and KM obtained by fitting the data to the Michaelis–Menten equation using an unweighted least-squares analysis (r2 ¼ 0:98).
Discussion This is the first report of rat monoclonal catalytic antibodies and the first report of monoclonal b-lactamase antibodies generated by immunisation with a transition-state analogue. For the important b-lactam substrate 3, a direct comparison may be made between the values obtained for the monoclonal catalytic antibodies at pH 9 and 37 °C and the published [15] values for the polyclonal catalytic antibodies under identical conditions. The monoclonal catalytic antibodies give a measured catalytic rate constant kcat ¼ 9:5 105 s1 , which is remarkably similar to the estimated catalytic rate constant kcat ¼ 1:6 104 s1 [15] for the catalytic fraction of the earlier sheep polyclonal preparation. However, since the estimate was based on the upper limit of the proportion of catalytic antibodies within the total IgG, it is probable that the polyclonal IgG contains catalytic antibodies of considerably greater activity. Neither the polyclonal nor the monoclonal antibodies were expected to catalyse the hydrolysis of the b-lactam, since the phosphate immunogen 1a was designed to generate antibodies that would catalyse the hydrolysis of carbonates such as 2a and 2b. The b-lactam 3 lacks major parts of the structural features expected to contribute to recognition and binding. In addition, the blactam ring is expected to present steric hindrance to binding and has N–C–O bond angles (in the ground state and transition state) that deviate from the equivalent O–C–O bond angles (in the carbonate ground state and transition state) and the O–P–O angle in the hapten. It is likely, therefore, considering the quantitative and qualitative similarity of the catalytic activities observed, that similar modes of binding may be operating in the
276
E.L. Ostler et al. / Biochemical and Biophysical Research Communications 299 (2002) 273–276
rat monoclonal antibody and the catalytic fraction of the sheep polyclonal antibody preparation. Only seven monoclonal antibodies (or antibody fragments) have been generated that catalyse the hydrolysis of amides. Of these, only two catalyse the hydrolysis of b-lactams and neither was generated by immunisation with a transition-state analogue. The results described above add one more monoclonal catalytic antibody and this was generated by using a transition-state analogue. We predict that analogues that are more closely related to the transition-state of blactam hydrolysis (for example, cyclic phosphonates or phosphonamidates) will generate monoclonal antibodies with greater b-lactamase activity. The earlier polyclonal antibody studies were valuable in uncovering b-lactamase activity where it was not expected. The monoclonal studies, described above, have confirmed the polyclonal results and demonstrated the similar properties of antibodies from different sources. Monoclonal antibodies are more amenable to structure determination and detailed investigations of the modes of b-lactam binding than are polyclonal antibodies. This avenue of investigation will be the subject of future experiments.
Acknowledgments We thank The Wellcome Trust for financial support (Grant reference 060295). Guillaume Boucher was supported by a TMR award (ERBFMRXCT 980 193) from the European Commission.
References [1] A. Tramontano, K.D. Janda, R.A. Lerner, Catalytic antibodies, Science 234 (1986) 1566–1570. [2] S.J. Pollack, J.W. Jacobs, P.G. Schultz, Selective chemical catalysis by an antibody, Science 234 (1986) 1570–1573. [3] R.A. Lerner, S.J. Benkovic, P.G. Schultz, At the crossroads of chemistry and immunology—catalytic antibodies, Science 252 (1991) 659–667. [4] J.D. Stevenson, N.R. Thomas, Catalytic antibodies and other biomimetic catalysts, Nat. Prod. Rep. 17 (2000) 535–577. [5] K.D. Janda, D. Schloeder, S.J. Benkovic, R.A. Lerner, Induction of an antibody that catalyzes the hydrolysis of an amide bond, Science 241 (1988) 1188–1191. [6] M.T. Martin, T.S. Angeles, R. Sugasawara, N.I. Aman, A.D. Napper, M.J. Darsley, R.I. Sanchez, P. Booth, R.C. Titmas, Antibody-catalyzed hydrolysis of an unsubstituted amide, J. Am. Chem. Soc. 116 (1994) 6508–6512.
[7] F. Benedetti, F. Berti, A. Colombatti, C. Ebert, P. Linda, F. Tonizzo, Anti-sulfonamide antibodies catalyse the hydrolysis of a heterocyclic amide, Chem. Commun. (1996) 1417–1418. [8] C.S. Gao, B.J. Lavey, C.H.L. Lo, A. Datta, P. Wentworth, K.D. Janda, Direct selection for catalysis from combinatorial antibody libraries using a boronic acid probe: primary amide bond hydrolysis, J. Am. Chem. Soc. 120 (1998) 2211–2217. [9] B. Gong, P.G. Schultz, S.S. Yoon, A catalytic antibody for the hydrolysis of peptides, J. Korean Chem. Soc. 42 (1998) 462–466. [10] B. Avalle, D. Thomas, A. Friboulet, Functional mimicry: elicitation of a monoclonal anti-idiotypic antibody hydrolyzing b-lactams, FASEB J. 12 (1998) 1055–1060. [11] F. Tanaka, H. Almer, R.A. Lerner, C.F. Barbas, Catalytic singlechain antibodies possessing b-lactamase activity selected from a phage displayed combinatorial library using a mechanism-based inhibitor, Tetrahedron Lett. 40 (1999) 8063–8066. [12] G.M. Blackburn, S.X. Deng, Catalytic antibodies for the hydrolysis of unactivated peptides, Biochem. Soc. Trans. 21 (1993) 1102–1107. [13] G. Gallacher, C.S. Jackson, M. Searcey, G.T. Badman, R. Goel, C.M. Topham, G.W. Mellor, K. Brocklehurst, A polyclonal antibody preparation with Michaelian catalytic properties, Biochem. J. 279 (1991) 871–881. [14] M. Resmini, R. Vigna, C. Simms, N.J. Barber, E.P. HagiPavli, A.B. Watts, C. Verma, G. Gallacher, K. Brocklehurst, Characterisation of the hydrolytic activity of a polyclonal catalytic antibody preparation by pH-dependence and chemical modification studies: evidence for the involvement of Tyr and Arg side chains as hydrogen-bond donors, Biochem. J. 326 (1997) 279–287. [15] E.L. Ostler, M. Resmini, G. Boucher, N. Romanov, K. Brocklehurst, G. Gallacher, Polyclonal antibody-catalysed hydrolysis of a b-lactam, Chem. Commun. (2002) 226–227. [16] H. Takahata, Y. Ohnishi, T. Yamazaki, A convenient synthesis of monocyclic b-lactams, Heterocycles 14 (4) (1980) 467–469. [17] C.J. Dean, Methods Mol. Biol. 10 (1992) 43–63. [18] M. Resmini, S. Gul, S. Carter, S. Sonkaria, C.M. Topham, G. Gallacher, K. Brocklehurst, A general kinetic approach to investigation of active-site availability in macromolecular catalysts, Biochem. J. 346 (2000) 117–125. [19] M. Stahl, B. Goldie, S.P. Bourne, N.R. Thomas, Catalytic antibodies generated by in-vitro immunization, J. Am. Chem. Soc. 117 (1995) 5164–5165. [20] G. Gallacher, C.S. Jackson, M. Searcey, R. Goel, G.W. Mellor, C.Z. Smith, K. Brocklehurst, Catalytic antibody-activity elicited by active immunization—evidence for natural variation involving preferential stabilization of the transition-state, Eur. J. Biochem. 214 (1993) 197–207. [21] G. Gallacher, M. Searcey, C.S. Jackson, K. Brocklehurst, Polyclonal antibody-catalyzed amide hydrolysis, Biochem. J. 284 (1992) 675–680. [22] G.M. Blackburn, J.D. Plackett, Strain effects in acyl transfer reactions. Part 1. The kinetics of hydrolysis of some N-aryl lactams, J. Chem. Soc., Perkin Trans. 2 (1973) 1366–1371. [23] T.J. Broxton, L.W. Deady, Mechanism of the basic methanolysis of benzanilides, J. Org. Chem. 39 (1974) 2767. [24] A.R. Butler, K.A. Freeman, D.E. Wright, A study of factors affecting the rates of hydrolysis of some N-aryl-azetidin-2-ones (Naryl-b-lactams), J. Chem. Soc., Perkin Trans. 2 (1977) 765–769.