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 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 pK(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 rho 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.
Aggarwal
Ranjana
R
Dipartimento di Scienze Chimiche Università degli Studi di Trieste Via Giorgieri 1, 34127 Trieste, Italy.
Benedetti
Fabio
F
Berti
Federico
F
Buchini
Sabrina
S
Colombatti
Alfonso
A
Dinon
Francesca
F
Galasso
Vinicio
V
Norbedo
Stefano
S
D000577:Amides / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D006433:Hemocyanins / Q000008:administration & dosage / Q000276:immunology; D006868:Hydrolysis; D051379:Mice; D008807:Mice, Inbred BALB C; D013237:Stereoisomerism
D000577:Amides; D000906:Antibodies; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin