A Catalytic Antibody Programmed for Torsional Activation of Amide Bond Hydrolysis

Amidase antibody 312d6, obtained against the sulfonamide hapten 4 a that mimics the transition state for hydrolysis of a distorted amide, accelerates the hydrolysis of the corresponding amides 1 a–3 a 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 1 a and 3 a shows a simple first-order dependence on [OH⁻], while hydrolysis of 3 a is zeroth-order in [OH⁻] below pH 8. The pH profile for hydrolysis of the corresponding tryptophan amide 2 a is more complex due to the dissociation of the zwitterion into an anion with pK<INF>a</INF> 9.74; hydrolysis of the zwitterionic and the anionic form of 2 a both show simple first-order dependence on [OH⁻]. Absence of ¹⁸O exchange between H<INF>2</INF>¹⁸O/¹⁸OH⁻ and the substrate, a normal SKIE for both 1 a (k<INF>H</INF>/k<INF>D</INF>=1.12) and 3 a (k<INF>H</INF>/k<INF>D</INF>=1.24) and the value of the Hammett constant ρ for hydrolysis of p-substituted amides 3 a–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 3 a 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 3 a 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 5 a–e confirm the high specificity of antibody 312d6 for p-methyl substituted substrates.