Premium
A Catalytic Antibody Programmed for Torsional Activation of Amide Bond Hydrolysis
Author(s) -
Aggarwal Ranjana,
Benedetti Fabio,
Berti Federico,
Buchini Sabrina,
Colombatti Alfonso,
Di Francesca,
Galasso Vinicio,
Norbedo Stefano
Publication year - 2003
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.200204620
Subject(s) - chemistry , hydrolysis , amide , amidase , medicinal chemistry , hydrolysis constant , nucleophile , catalysis , tetrahedral carbonyl addition compound , zwitterion , amine gas treating , peptide bond , reaction rate constant , stereochemistry , organic chemistry , kinetics , molecule , enzyme , physics , quantum mechanics
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 10 3 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 p K a 9.74; hydrolysis of the zwitterionic and the anionic form of 2 a both show simple first‐order dependence on [OH − ]. Absence of 18 O exchange between H 2 18 O/ 18 OH − and the substrate, a normal SKIE for both 1 a ( k H / k D =1.12) and 3 a ( k H / k D =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.