Efficient and Selective P‐nitrophenyl‐ester‐hydrolyzing Antibodies Elicited by a P‐nitrobenzyl Phosphonate Hapten (original) (raw)
J Mol Biol, 1998
The antibody D2.3 catalyzes the hydrolysis of several p-nitrobenzyl and p-nitrophenyl esters with signi®cant rate enhancement; product inhibition is observed with the former compounds but not with the latter. Whereas enzyme speci®city has been extensively studied by X-ray crystallography, structural data on catalytic antibodies have thus far related only to one of the reactions they catalyze. To investigate the substrate speci®city and the substrate relative to product selectivity of D2.3, we have determined the structures of its complexes with two p-nitrophenyl phosphonate transition state analogs (TSAs) and with the reaction product, p-nitrophenol. The complexes with these TSAs, determined at 1.9 A Ê resolution, and that with p-nitrobenzyl phosphonate determined previously, differ mainly by the locations and conformations of the ligands. Taken together with kinetic data, the structures suggest that a hydrogen bond to an atom of the substrate distant by eight covalent bonds from the carbonyl group of the hydrolyzed ester bond contributes to catalytic ef®ciency and substrate speci®city. The structure of Fab D2.3 complexed with p-nitrophenol was determined at 2.1 A Ê resolution. Release of p-nitrophenol is facilitated due to the unfavourable interaction of the partial charge of the nitro group of p-nitrophenolate with the hydrophobic cavity where it is located, and to the absence of a direct hydrogen bond between the product and the Fab. Catalytic speci®city and the manner of product release are both affected by interactions with substrate atoms remote from the reaction center that were not programmed in the design of the TSA used to elicit this antibody. Selection of a catalytic antibody that makes use of TSA unprogrammed features has been made practical because of the screening for catalytic ef®ciency incorporated in the procedure used to obtain it.
Mechanism of Inactivation of a Catalytic Antibody by P-Nitrophenyl Esters
European Journal of Biochemistry, 1997
Antibody CNJ206 catalyses the hydrolysis of p-nitrophenyl esters with significant rate enhancement; however, after a few cycles, 90% of the catalytic activity of CNJ206 is irreversibly lost. This report investigates the properties of the inactivated Fab (fragment antigen binding). After inactivation, the residual esterase activity of CNJ206 is similar to that of the catalytic antibody inhibited by the transition-state analogue (TSA) used to elicit it; the affinity of CNJ206 for the TSA is also dramatically lowered. Here we propose a simple scheme that accounts for the steady-state kinetics of inactivation. The following lines of evidence, when taken together, suggest that stable acylated tyrosine side chains within or close to the Fab combining site are involved in the inactivation process: isoelectric focusing and matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometry show that incubation with substrate results in several acylated Fab species; inactivation is stable at pH 8, is reversed by mild hydroxylamine treatment and follows the same kinetics as inhibition of binding, which is slowed down by the presence of the TSA hapten. Analysis of the Fab-TSA X-ray structure shows that three tyrosine residues are potential candidates for the inactivation of CNJ206 by its substrates, Tyr L96 being the most likely one; this also suggests that site-directed mutation of one or more of these residues might prevent substrate inactivation and significantly improve catalysis.
Simple method for selecting catalytic monoclonal antibodies that exhibit turnover and specificity
Biochemistry, 1990
Monoclonal antibodies were raised against a mono-p-nitrophenyl phosphonate ester to elicit catalytic antibodies capable of hydrolyzing the analogous p-nitrophenyl ester or carbonate. Potential catalytic antibody producing clones were selected, by use of a competitive inhibition assay, on the basis of their affinity for a "short" transition-state analogue, a truncated hapten which maximizes the relative contribution of the transition-state structural elements to binding. Of 30-40 clones that would have been examined on the basis of hapten binding alone, 7 were selected and 4 of these catalyzed the hydrolysis of the relevant p-nitrophenyl ester. This competitive inhibition technique represents a general approach for selecting potential catalytic antibodies and significantly increases the probability of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (kcat/kuncat-lo4) and substrate specificity for the hydrolysis of the analogous ester and, for three of the antibodies, of the analogous carbonate. The antibodies displayed turnover, an essential feature of enzymes. Evidence that catalysis occurred at the antibody combining sites was provided by the identity of the binding and the catalysis-Inhibition specificity patterns.
Crossreactivity, efficiency and catalytic specificity of an esterase-like antibody
Journal of Molecular Biology, 1998
The antibody D2.3 catalyzes the hydrolysis of several p-nitrobenzyl and p-nitrophenyl esters with signi®cant rate enhancement; product inhibition is observed with the former compounds but not with the latter. Whereas enzyme speci®city has been extensively studied by X-ray crystallography, structural data on catalytic antibodies have thus far related only to one of the reactions they catalyze. To investigate the substrate speci®city and the substrate relative to product selectivity of D2.3, we have determined the structures of its complexes with two p-nitrophenyl phosphonate transition state analogs (TSAs) and with the reaction product, p-nitrophenol. The complexes with these TSAs, determined at 1.9 A Ê resolution, and that with p-nitrobenzyl phosphonate determined previously, differ mainly by the locations and conformations of the ligands. Taken together with kinetic data, the structures suggest that a hydrogen bond to an atom of the substrate distant by eight covalent bonds from the carbonyl group of the hydrolyzed ester bond contributes to catalytic ef®ciency and substrate speci®city. The structure of Fab D2.3 complexed with p-nitrophenol was determined at 2.1 A Ê resolution. Release of p-nitrophenol is facilitated due to the unfavourable interaction of the partial charge of the nitro group of p-nitrophenolate with the hydrophobic cavity where it is located, and to the absence of a direct hydrogen bond between the product and the Fab. Catalytic speci®city and the manner of product release are both affected by interactions with substrate atoms remote from the reaction center that were not programmed in the design of the TSA used to elicit this antibody. Selection of a catalytic antibody that makes use of TSA unprogrammed features has been made practical because of the screening for catalytic ef®ciency incorporated in the procedure used to obtain it.
Enantioselective hydrolysis of naproxen ethyl ester catalyzed by polyclonal antibodies
Chinese Science Bulletin, 1997
BASED on the hypothesis of ~a u l i n~[ ' ] on enzyme action that free energy difference between the ground and transition state is reduced by the specific binding of active center of enzyme to the intermediate, a large number of antibodies with catalytic power have been obtained successively by using designed molecules (mimicking the stereoelectronic features of the transition state for the reactions) as haptens conjugated to carrier proteins to immunize the animals and screen out the desired monoclonal antibodies through hy bridoma technology[2' 31. The catalytic antibodies which combined the tremendous diversity of antibodies with catalytic power of enzyme opened new horizons of chemical and immunological fields. Antibodies that catalyze a variety of additional reactions, including all types of reactions catalyzed by enzyme, for example,
Regioselective nitration of phenol induced by catalytic antibodies
Journal of protein chemistry, 2002
Catalytic antibodies with a metalloporphyrin cofactor represent a new generation of biocatalysts tailored for selective oxidations. Thus monoclonal antibodies, 3A3, were raised against microperoxidase 8 (MP8), and the corresponding 3A3-MP8 complexes were shown previously to have a high peroxidase activity. This paper shows that those complexes also catalyzed efficiently the nitration of phenol into 2- and 4-nitrophenol by NO2- in the presence of H2O2. pH dependence studies suggested that no amino acid from the antibody protein participated in the heterolytic cleavage of the O-O bond of H2O2. The inhibition of the reaction by cyanide and radical scavengers suggested a MP8-mediated peroxidase-like mechanism, involving the reduction of high-valent iron-oxo species by NO2- and phenol producing, respectively, NO2* and phenoxy radicals, which then reacted to give nitrophenols. Finally, the antibody protein appears to have two major roles: (i) it protects MP8 toward oxidative degradations ...
Effective heterogeneous hydrolysis of phosphodiester by pyridine-containing metallopolymers
Inorganica Chimica Acta, 2005
The copper (II) complex of a simple pyridine-and amide-containing copolymer serves as an effective catalyst for heterogeneous hydrolysis of the prototypical phosphodiester substrate bis(p-nitrophenyl)phosphate at pH 8.0 and 25 °C. The catalysis has a firstorder rate constant of k cat = 8.3 • 10 À6 s À1 , corresponding to a catalytic proficiency of 75-thousand folds relative to the uncatalyzed hydrolysis with a rate constant of k 0 = 1.1 • 10 À10 s À1 in aqueous buffer solution at pH 8.0. This observation suggests that polymers can be designed to include various functional groups feasible for effective metal-centered catalysis of phosphodiester hydrolysis.