Amidase antibody 312d6, obtained against the sulfonamide hapten 4a that mimics the transition state for hydrolysis of a distorted amide, accelerates the hydrolysis of the corresponding amides 1a-3a by a factor of 103 at pH 8. The mechanisms of both the uncatalyzed and antibody-catalyzed reactions were studied. Between pH 8 and 12 the uncatalyzed hydrolysis of N-toluoylindoles 1a and 3a shows a simple first-order dependence on [OH-], while hydrolysis of 3a is zeroth-order in [OH-] below pH 8. The pH profile for hydrolysis of the corresponding tryptophan amide 2a is more complex due to the dissociation of the zwitterion into an anion with pKa 9.74; hydrolysis of the zwitterionic and the anionic form of 2a both show simple first-order dependence on [OH-]. Absence of 18O exchange between H2 18O/18OH- and the substrate, a normal SKIE for both 1a (kH/kD = 1.12) and 3a (kH/kD = 1.24) and the value of the Hammett constant ρ for hydrolysis of p-substituted amides 3a-e are consistent with an ester-like mechanism in which formation of the tetrahedral intermediate is rate-determining and the amine departs as anion. The 312d6-catalyzed hydrolysis of 3a was studied between pH 7.5 and 9, and its independence of pH in this range indicates that water is the reacting nucleophile. Hydrolysis of 3a is only partially inhibited by the sulfonamide hapten, and this indicates that non-specific catalysis by the protein accompanies the specific process. Only the nonspecific process is observed in the hydrolysis of amides 3 with para substituents other than methyl. Binding studies on the corresponding series of p-substituted sulfonamides 5a-e confirm the high specificity of antibody 312d6 for p-methyl substituted substrates.
- Catalytic antibodies
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