Document Type
Article
Publication Date
8-2-1994
Publication Title
Proceedings of the National Academy of Sciences of the United States of America
Abstract
Antibody 43C9 accelerates the hydrolysis of a p-nitroanilide by a factor of 2.5 x 10(5) over the background rate in addition to catalyzing the hydrolysis of a series of aromatic esters. Since this represents one of the largest rate accelerations achieved with an antibody, we have undertaken a series of studies aimed at uncovering the catalytic mechanism of 43C9. The immunogen, a phosphonamidate, was designed to mimic the geometric and electronic characteristics of the tetrahedral intermediate that forms upon nucleophilic attack by hydroxide on the amide substrate. Further studies, however, revealed that the catalytic mechanism is more complex and involves the fortuitous formation of a covalent acyl-antibody intermediate as a consequence of complementary side chain residues at the antibody-binding site. Several lines of evidence indicate that the catalytic mechanism involves two key residues: His-L91, which acts as a nucleophile to form the acyl-antibody intermediate, and Arg-L96, which stabilizes the anionic tetrahedral moieties. Support for this mechanism derives from the results of site-directed mutagenesis experiments and solvent deuterium isotope effects as well as direct detection of the acyl-antibody by electrospray mass spectrometry. Despite its partial recapitulation of the course of action of enzymic counterparts, the reactivity of 43C9, like other antibodies, is apparently limited by its affinity for the inducing immunogen. To go beyond this level, one must introduce additional catalytic functionality, particularly general acid-base catalysis, through either improvements in transition-state analog design or site-specific mutagenesis.
Recommended Citation
Stewart, Jon D.; Krebs, Joseph F.; Siuzdak, Gary; Berdis, Anthony J.; Smithrud, David B.; and Benkovic, Stephen J., "Dissection of An Antibody-Catalyzed Reaction" (1994). Chemistry Faculty Publications. 184.
https://engagedscholarship.csuohio.edu/scichem_facpub/184
Version
Postprint
Publisher's Statement
© 1994 by The National Academy of Sciences
Volume
91
Issue
16
Comments
This work was partially supported by grants from the Office Of Naval Research(N00014-91-J-1593),A postdoctoral fellowship from the Helen Hay Whitney Foundation (J.D.S.), and a postdoctoral fellowship from the National Institutes Of Health(D.B.S.;GM 15536-02).