A Catalytic Antibody Programmed for Torsional Activation of Amide Bond Hydrolysis

摘要

Amidase antibody 312d6 obtained against the sulfonamide hapten 4a that mimics the transitin state for hydrolysis of distrted amide accelerates the hydrolysis of the corresponding amides 1a-3a by a factor of 10~3 at pH 8 The mechanisms of tboth the uncatalyzed and antibody-catalyzed reactions were studied Between pH 8 and 12 the uncatalyzed hydrolysis of N-toluoylindoles 1a and 3a shows a smiple 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 zwitterions into an anion with pK_a 9.74 hydrolysis of the zwittrionic and the anionic form of 2a both show simple first-order dependence on [OH~-] Absence of ~18O excfhange be tween H_2~18O/~18OH~- and the substrate a mormal SKIE for both 1a (k_H/k_D=1.12) and 3a (k_H/k_D=1.24) and the value of the Hammett constant rho for hydrolysis of p-substitute amides 3a-e are consistent with an ester-like mechanism in whicl formation of ht tetrahedral intermediate is rate-determining and the amine departs as anion The 312d6-catalyzed hydrolysis of 3a waa studied between pH 7.5 and 9 and its independence of pH in this range indicates that water is the reactin nucleophile Hy droglysis of 3a is only partially inhibited by the sulfonamide hapten and this indicates that non-specific acatalysis by the protein accompanies the apecific process only the nonspecific process is observed in the hydroglysis fo amides 3 with para substitutent other than methyl BGinding studies on the corresponding series of p-substituted sulfonamides 5a-e confirm the highe specificity of antibody 312d6 for p-methyl subgstituted substrates.