Proc. Natl. Acad. Sci. USA Vol. 85, pp. 4953-4955, July 1988 Chemistry Catalysis of concerted reactions by antibodies: The Claisen rearrangement (transition state analog/monoclonal antibody/chorismate mutase model) DONALD HILVERT*, STEPHEN H. CARPENTER, KAREN D. NARED, AND MARIA-TERESA M. AUDITOR Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037 Communicated by Emil Thomas Kaiser, March 28, 1988 (receivedfor review March 15, 1988)

ABSTRACT Monoclonal antibodies were prepared against The oxabicyclic compound 4a, which mimics the putative a transition state analog inhibitor of chorismate mutase (EC transition state structure, is the best known inhibitor of 5.4.99.5). One ofthe antibodies catalyzes the rearrangement of chorismate mutase (13). It binds approximately 100 times chorismate to prephenate with rate accelerations of more than more tightly to the enzyme than does chorismate (13). We 2 orders of magnitude compared to the uncatalyzed reaction. have synthesized a derivative of4a and used it as a hapten to Saturation kinetics were observed, and at 250C the values of elicit monoclonal antibodies. Having completed a prelimi- k,.t and Km were 1.2 x 1O-3 S-' and 5.1 x 10-5 M respec- nary screen of 15 ofthe 46 resulting antibodies, we report that tively. The transition state analog was shown to be a compet- one of these significantly accelerates the rearrangement of itive inhibitor of the reaction with Ki equal to 0.6 JIM. These chorismate to prephenate. results demonstrate the feasibility of using rationally designed immunogens to generate antibodies that catalyze concerted MATERIALS AND METHODS reactions. Instrumentation. UV-visible spectra were measured on a Perkin-Elmer Lambda-4B spectrophotometer equipped with Recent experiments have utilized the immune system as a a thermostatically regulated cell holder. High-performance prolific source of specific receptor molecules for the con- liquid chromatography (HPLC) was performed with a Hitachi struction of enzyme-like catalysts (1-3). Using the transition model 655A-11 liquid chromatography system with a variable state analog approach enunciated by Jencks in 1969 (4), two wavelength detector and an analytical Vydac C18 218-TP-54 groups of researchers elicited antibodies against aryl phos- reverse-phase column (The Separations Group, Hesperia, phonate esters that catalyze the cleavage of structurally CA). NMR spectra were obtained on a Bruker NMR spec- homologous aryl esters (1, 3) and carbonates (2). In principle, trometer (300 MHz). catalysis results when binding interactions stabilize the Materials. (-)-Chorismic acid (80-90%) was purchased transition state structure of a particular reaction relative to from Sigma as the free acid and used without further the bound ground state. Given the enormous specificity purification. The concentrations of stock solutions of choris- inherent in antibody binding interactions, and the fact that mate were determined by UV spectroscopy (E275, 2630 M -' antibodies can be generated against virtually any substance, cm 1) (11). The oxabicyclic transition state analog 4a was it may be possible to prepare tailored catalysts for many synthesized according to published procedures (13), together chemical transformations with this strategy. with its exo epimer (1:5 mixture, endo/exo). The desired Since there is presumably a low probability of generating endo isomer was purified as the bis(trimethylsilylethyl) ester an effective constellation of multiple catalytic groups (e.g., by repeated flash chromatography and deprotected, as de- general acids, general bases, nucleophiles) in the binding site scribed (13). A glutaric acid linker was used to couple the of an antibody during the immunization event, reactions that hapten to the carrier proteins keyhole limpet hemocyanin do not require chemical catalysis are perhaps more suitable (KLH, Pacific Bio Laboratories, Venice, CA) and bovine for development. Hence, we have begun to prepare antibod- serum albumin (BSA, Sigma). The coupling agent 4b was ies that catalyze concerted reactions, including Claisen re- prepared in high yield from the bis(trimethylsilylethyl) ester arrangements and Diels-Alder These processes of 4a by a two-step procedure involving acylation of the cyclizations. secondary hydroxyl group with the mono acid chloride, mono are expected to be sensitive to the principal catalytic effects N-hydroxysuccinimide ester of glutaric acid in the presence antibodies are likely to impart: induced strain and proximity of triethylamine, followed by deprotection with tetrabutyl- (5). Moreover, they are important transformations available ammonium fluoride or trifluoroacetic acid immediately be- to organic chemists for the synthesis of biologically active fore use. All compounds gave satisfactory NMR spectra. molecules (6). Treatment of the carrier proteins with a 100-fold excess of 4b The conversion of chorismate (Scheme I, structure 1) into in 10 mM borate buffer, pH 9/0.2 M NaCl for 1-2 hr at room prephenate (structure 2) is an example of a biologically temperature, followed by gel filtration on Sephadex G-50, relevant Claisen rearrangement. It is the key transformation gave the desired protein conjugates. Approximately 20-30 in the biosynthesis ofaromatic amino acids in bacteria, fungi, molecules of 4 could be attached to KLH and BSA by using and higher plants (7). The enzyme chorismate mutase (EC this protocol, as estimated by amine titration (14). 5.4.99.5) accelerates this reaction 2 x 106-fold over the Preparation and Purification of Monoclonal Antibodies. uncatalyzed thermal process (8). Although the precise mech- Mice (129 GIX+ strain) were immunized with the KLH anism of rearrangement is still contested (9-11), elegant conjugate 4c emulsified in complete Freund's adjuvant (15). stereochemical studies have implicated a transition state with Serum titer was determined with the BSA conjugate by an diaxial chairlike geometry (structure 3) (12). enzyme-linked immunosorbent assay (ELISA) (16). A mouse

The publication costs of this article were defrayed in part by page charge Abbreviations: BSA, bovine serum albumin; KLH, keyhole limpet payment. This article must therefore be hereby marked "advertisement" hemocyanin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 4953 Downloaded by guest on September 24, 2021 4954 Chemistry: Hilvert et al. Proc. Natl. Acad. Sci. USA 85 (1988)

K 00C COO- 000, COO- ' a cooH

OH OH OH

3 I 2

H

= -OOoc 4a R H 0

-Coo0 4b R = O-hl

0 4c R = NHFKLM

0 0 Scheme I

with a serum titer (the dilution at which 50% of the available hapten-specific immunoglobulins were identified from a sin- ligand is bound to antibody) of 1:6400 was used to obtain gle fusion and cloned. Monoclonal antibodies were obtained hybridomas by fusion of spleen cells with SP2/0 + myeloma from ascites fluid and purified to at least 90% homogeneity by cells according to standard protocols (15, 16). Hybridoma ion-exchange chromatography (16). To date, 15 different IgG cells that secreted IgG antibodies specific for the hapten were antibodies have been tested for their ability to catalyze the cloned and subsequently propagated in (BALB/c x 129 Claisen rearrangement of chorismate to prephenate. Four- GIX+)F1 mouse ascites (15, 16). teen of these had little effect on the reaction rate, measured Monoclonal antibodies were obtained from mouse ascites spectroscopically at 275 nm, even though they bound the by ammonium sulfate precipitation followed by chromatog- BSA conjugate of 4a tightly. Antibody 1F7, on the other raphy on DEAE-Sephacel (16). Protein was eluted from the hand, showed considerable activity in the preliminary assay gel with a NaCI step gradient (0, 50, 100, 150, 250, 500 mM) and was selected for further characterization. in 10 mM Tris-HCl (pH 8.0). IgG typically was eluted at 100 To establish the nature of the catalyzed reaction, the mM NaCl and was concentrated and dialyzed. Antibodies following experiments were performed: (i) the rate of disap- were to pure judged be 90%o by Coomassie blue staining after pearance ofchorismate was determined by HPLC and shown was NaDodSO4/PAGE. Immunoglobulin isotype determined to be within experimental error of the spectroscopically with a Fisher Clonotyping kit. Protein concentrations were determined rate; (ii) prephenate was identified as the product estimated by the method of Smith et al. (17). IgG molecular of the reaction by conversion to phenylpyruvate in acid and weight was assumed to be 160,000 (18). quantification of the enol in alkaline solution at 320 nm (20); Antibody Assays. Antibodies were assayed for chorismate mutase activity in 10 mM Tris HCl, pH 8.0/100 mM NaCl at (iii) the rate of prephenate formation in the presence of 1F7 was shown to correlate directly with the rate of chorismate constant temperature. The disappearance of chorismate was and (iv) was to be first- followed by UV spectroscopy at 275 nm and by reversed- disappearance; the reaction shown phase HPLC. HPLC-response factors were determined by order with respect to antibody. using authentic standards; p-aminobenzoic acid served as an When initial rates with 1F7 were measured as a function of internal standard for HPLC kinetics. Initial rates were chorismate concentration, saturation kinetics were ob- determined from the first 5-10% of reaction for a range of served. This finding is consistent with formation of a Mi- substrate concentrations bracketing the Km value and were chaelis complex between the substrate and the antibody prior corrected for background activity. Rates determined by the to rearrangement, as shown in the following scheme: two methods were the same within 10%. The kinetic con- ki kw stants were obtained by direct computer fit of the data to the 1F7 + 1 1F7-1 -0 1F7 + 2 Michaelis-Menten equation: vo = kcat2[AbI[SI/(Km + [SI) (19), where [Ab] is the antibody concentration and [S] is the At 25°C the values of kcat and Km were determined to be 1.2 substrate concentration. X 10-3 s-' and 5.1 x 10' M, respectively (Table 1). The concentration of prephenate was quantitated by con- Comparison of the kcat value with the rate constant for the version to phenylpyruvate under acid conditions and mea- uncatalyzed thermal rearrangement at this temperature (kun) surement of the absorbance of the enol in alkaline solution gives a 190-fold rate acceleration per binding site. (6320, 17,000 M-' cm-1) (20). The rate of formation of pre- Convincing evidence that chorismate reacts in the induced phenate was estimated by performing this assay on aliquots binding pocket of the antibody was obtained by examining of a reaction mixture at specific time intervals. the kinetics in the presence of the transition state analog. As shown in Fig. 1, the conversion of chorismate to prephenate AND RESULTS DISCUSSION is competitively inhibited by 4a, with a Ki value of -0.6 x Monoclonal antibodies were raised against 4c by standard 10 -6 M. The ratio ofK; to Km is similar to that seen in the case techniques (15, 16). Forty-six hybridomas that secreted of chorismate mutase itself (13). Moreover, it correlates Downloaded by guest on September 24, 2021 Chemistry: Hilvert et al. Proc. Natl. Acad. Sci. USA 85 (1988) 4955 Table 1. Kinetic parameters for the rearrangement of chorismate The Claisen rearrangement of chorismate is a prototype of to prephenate catalyzed by monoclonal antibody 1F7 a broad class of concerted chemical reactions of enormous Temp., kcat x 103, Km x 105, kcat/Km9 practical and theoretical interest. Using a rationally designed °C s - l M M '-s-l kcat/kun immunogen, we have successfully elicited an antibody that significantly accelerates the rate of this process. The design 14.0 0.39 ± 0.04 4.9 ± 0.8 8.0 ± 1.4 250 principles outlined above can now be extended to the 25.0 1.2 ± 0.2 5.1 ±0.9 24 ± 5 190 development of other antibodies that are shape-selective 36.0 2.5 ± 0.2 3.8 ±0.4 66 ± 5 110 rather than chemoselective. The ability to generate tailored Parameters were determined by fitting initial-rate data, obtained at catalysts for these important transformations has far- 275 nm in 10 mM Tris HCl, pH 8.0/100 mM NaCl, to the Michaelis- reaching implications for medicine and industry. Menten equation. The values of kung the rate constant for the uncatalyzed thermal rearrangement, were extrapolated from the data Note Added in Proof. Recent experiments with (± )-chorismate have of Andrews et al. (8). established that the antibody-catalyzed rearrangement not only is accelerated over background but also occurs in a highly stereose- roughly (within a factor of 2) with the observed rate accel- lective fashion. (+)-Chorismate, unlike the (-) isomer, does not eration, consistent with the notion that the enhancement is appear to be a substrate for 1F7, demonstrating further the enzyme- due to increased binding of the transition state by the anti- like nature of this tailored antibody binding site. body. It is interesting that the dissociation constant measured We are grateful to Diane Schloeder for expert technical advice and for 4a, which contains an unacylated hydroxyl group, is 1-2 to Drs. Darryl Rideout and Alan Schwabacher for a critical reading orders of magnitude larger than that for the hapten linked to of the manuscript. This work was supported in part by a Junior BSA [estimated by ELISA titration (21)]. Since binding Faculty Research Award to D.H. from the American Cancer Society interactions distant from the reaction center can contribute and by Grant GM38273 from the National Institutes of Health to significantly to the reaction rate (22), an acylated chorismate D.H. derivative may be an even better substrate for the antibody. 1. Tramontano, A., Janda, K. D. & Lerner, R. A (1986) Science To gain some insight into the mechanism of catalysis by 234, 1566-1570. 1F7, the chorismate rearrangement was examined at two 2. Jacobs, J. W., Schultz, P. G., Sugasawara, R. & Powell, M. additional temperatures. The relevant kinetic parameters are (1987) J. Am. Chem. Soc. 109, 2174-2176. summarized in Table 1. The entropy and enthalpy of activa- 3. Napper, A. D., Benkovic, S. J., Tramontano, A. & Lerner, tion (ASt and AHt) for conversion of bound substrate to R. A. (1987) Science 237, 1041-1043. product were calculated from the temperature dependence of 4. Jencks, W. P. (1969) Catalysis in Chemistry and Enzymology (McGraw Hill, New York), p. 288. kcat to be - 22 + 6 cal-mol -K-1 and 15 + 2 kcal/mol (1 5. Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, kcal = 4184 J) respectively. For comparison, ASt is - 12.85 219-410. ± 0.42 cal mol-'K1-'and Ali is 20.71 ± 0.35 kcal/mol for 6. March, J. (1985) Advanced Organic Chemistry (Wiley, New the thermal conversion of chorismate to prephenate (8). It is York), pp. 745-758, 1028-1032. apparent that the antibody stabilizes the transition state for 7. Weiss, U. & Edwards, J. M. (1980) The Biosynthesis of rearrangement by significantly lowering the enthalpy of Aromatic Amino Compounds (Wiley, New York), pp. 134-184. activation. This might be accomplished through the use of 8. Andrews, P. R., Smith, G. D. & Young, 1. G. (1973) Biochem- binding energy to induce strain into the starting material (5). istry 18, 3492-3498. 9. Copley, S. D. & Knowles, J. R. (1987) J. Am. Chem. Soc. 109, The less favorable ASt value for the catalyzed reaction, on 5008-5013. the other hand, may reflect the need for a conformational 10. Gajewski, J. J., Jurayj, J., Kimbrough, D. R., Gande, M. E., change in the antibody combining site during reaction. Ganem, B. & Carpenter, B. K. (1987) J. Am. Chem. Soc. 109, 1170-1186. 200 11. Addadi, L., Jaffe, E. K. & Knowles, J. R. (1984) Biochemistry 22, 4494-4501. 12. Sogo, S. G., Widlanski, T. S., Hoare, J. H., Grimshaw, C. E., Berchthold, G. A. & Knowles, J. R. (1984) J. Am. Chem. Soc. 106, 2701-2703. 13. Bartlett, P. A. & Johnson, C. R. (1985) J. Am. Chem. Soc. 107, 7792-7793. 14. Habeeb, A. F. S. A. (1966) Anal. Biochem. 14, 328-336. 15. Niman, H. L. & Elder, J. H. (1980) Proc. Natl. Acad. Sci. USA 77, 4524-4528. 16. Goding, J. W. (1983) Monoclonal Antibodies: Principles and .0 Practice (Academic, New York), pp. 234-255. 17. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. & Klenk, D. C. (1985) Anal. Biochem. 150, 76-85. 18. Kabat, E. A. (1976) Structural Concepts in Immunology and Immunochemistry (Holt, Reinhart and Winston, New York), p. 0 lo 20 30 227. 19. Yamaoka, K., Tanigawara, Y., Nakagawa, T. & Uno, T. (1981) 1/[chorismate] (mM-1) J. Pharmacobio. Dyn. 4, 879-885. 20. Gorisch, H. & Lingens, F. (1974) Biochemistry 13, 3790-3794. FIG. 1. Lineweaver-Burk plot for Claisen rearrangement of 21. Butler, J. E. (1980) In Enzyme-Immunoassay, ed. Maggio, chorismate by monoclonal antibody 1F7. Velocities were determined E. T. (CRC, Boca Raton, FL), pp. 41-42. as described in Table 1. x, No inhibitor present; *, inhibited by 6.4 22. Moore, S. A. & Jencks, W. P. (1982) J. Biol. Chem. 257, ,uM 4a; c, inhibited by 15 uM 4a. 10893-10907. Downloaded by guest on September 24, 2021