The antibody 13G5 binds 6-Glutaramide benzisoxazole with low nanomolar affinity. 13G5 promotes the selective cleavage of 6-glutaramidebenzisoxazoles an unactivated substrateswith multiple turnovers. 13G5 exhibits a bell-shaped pH-rate profile, raising the possibility that bifunctional acid-base catalysis may be operative.
Figure 1 Brønsted (leaving group) plot for the decomposition of substituted benzisoxazoles catalyzed by antibodies 13G5 (○) and 47C4(●).
Initial velocities were determined at 20°C with substituted benzisoxazoles in the presence of the antibodies in phosphate buffer, and corrected for the rate of the background reaction under the same buffer conditions.
Kikuchi, K., Hannak, R. B., Guo, M. J., Kirby, A. J., & Hilvert, D. (2006). Toward bifunctional antibody catalysis. Bioorganic & medicinal chemistry, 14(18), 6189-6196.
Figure 2 Michaelis-Menten plot for the conversion of 6-glutaramide benzisoxazole 9 with 13G5.
At pH 7.4 and 20°C, saturation kinetics were observed, consistent with a kinetic mechanism in which substrate and antibody form a Michaelis-Menten complex before proton abstraction.
Kikuchi, K., Hannak, R. B., Guo, M. J., Kirby, A. J., & Hilvert, D. (2006). Toward bifunctional antibody catalysis. Bioorganic & medicinal chemistry, 14(18), 6189-6196.
Figure 3 Plots of kcat/Km versus pH for the 47C4-catalyzed elimination of 6-nitrobenzisoxazole (●) and the 13G5-catalyzed elimination of 5-nitrobenzisoxazole (○).
Kinetics were performed at 20°C in the following buffers (40 mM, containing 100 mM NaCl): sodium acetate (pH <6); sodium phosphate (6 < pH < 8); and sodium carbonate (pH>8).
Kikuchi, K., Hannak, R. B., Guo, M. J., Kirby, A. J., & Hilvert, D. (2006). Toward bifunctional antibody catalysis. Bioorganic & medicinal chemistry, 14(18), 6189-6196.
Figure 4 Plot of kcat versus pH for the 13G5-catalyzed elimination of 6-glutaramidebenzisoxazole 9.
Kinetics were performed at 20°C in the following buffers (40 mM, containing 100 mM NaCl): sodium acetate (pH <6); sodium phosphate (6 < pH < 8); and sodium carbonate (pH>8). Initial rates were determined spectrophotometrically as a function of time.
Kikuchi, K., Hannak, R. B., Guo, M. J., Kirby, A. J., & Hilvert, D. (2006). Toward bifunctional antibody catalysis. Bioorganic & medicinal chemistry, 14(18), 6189-6196.
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CAT | Product Name | Application | Type |
---|---|---|---|
HPAB-S0003-YC-F(E) | Mouse Anti-6-Glutaramide benzisoxazole Recombinant Antibody (clone 13G5); Fab Fragment | Catalyze | Mouse Fab |
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