Chemical equilibrium at an antibody binding site: catalytic efficiency defined by a Haldane relationship.
This paper describes the first study of a reaction catalyzed in both forward and reverse directions by an antibody. The rates of reversible addition of sulfite to 6-hydroxy-3H-xanthen-3-one (1) and their pH dependence are influenced by a monoclonal antibody to a fluorescein hapten. The antibody is presumed to recognize the two substrates in a chemically productive prereaction complex with a geometry similar to that between the carboxyl group and the xanthenyl group in fluorescein. Equilibria are determined for the reactions in solution and at the antibody combining site from rate constants for the forward and reverse reactions. The solution equilibrium lies in favor of adduct with KA = x 10(7) M-1 at pH 7.5. The corresponding equilibrium at the combining site is much smaller (KAb = 8), and this is attributed to the preferential binding of substrates 1 and sulfite. The magnitude of differential stabilization is determined from the equilibrium constants (2 x 10(6) M-1). Observed rates at low molar standard state are compared to reveal the acceleration of the process at the antibody combining site versus the bimolecular solution reaction. In the associative direction, the rate factor of 1200 may largely be attributed to the entropy savings for the reaction on the catalyst. This analysis serves to illustrate the potential for catalysis by antibodies due from recognition of structural differences between substrates of different energy on a reaction coordinate and suggests a strategy for inducing antibody catalysts that does not presume knowledge of catalytic mechanisms or transition-state structure.
Janjić
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Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Tramontano
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A
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002384:Catalysis; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D013447:Sulfites / Q000378:metabolism*; D014966:Xanthenes / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D013447:Sulfites; D014966:Xanthenes; C066954:6-hydroxy-3-xanthen-3-one