A Catalytic Antibody Programmed for Torsional Activation of Amide Bond Hydrolysis

Amidase antibody 312d6, obtained against the sulfonamide hapten 4 athat mimics the transition state for hydrolysis of a distorted amide, accelerates the hydrolysis of the corresponding amides 1 a–3 aby a factor of 103at 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 aand 3 ashows a simple first‐order dependence on [OH−], while hydrolysis of 3 ais zeroth‐order in [OH−] below pH 8. The pH profile for hydrolysis of the corresponding tryptophan amide 2 ais more complex due to the dissociation of the zwitterion into an anion with pKa9.74; hydrolysis of the zwitterionic and the anionic form of 2 aboth show simple first‐order dependence on [OH−]. Absence of 18O exchange between H218O/18OH−and the substrate, a normal SKIE for both 1 a(kH/kD=1.12) and 3 a(kH/kD=1.24) and the value of the Hammett constant ρfor hydrolysis of p‐substituted amides 3 a–eare 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 awas 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 ais 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 3with parasubstituents other than methyl. Binding studies on the corresponding series of p‐substituted sulfonamides 5 a–econfirm the high specificity of antibody 312d6 for p‐methyl substituted substrates.