Efficient and Selective <i>P</i>‐nitrophenyl‐ester‐hydrolyzing Antibodies Elicited by a <i>P</i>‐nitrobenzyl Phosphonate Hapten

Tawfik, Dan S.; Lindner, Ariel B.; Chap, Rachel; Eshhar, Zelig; Green, Bernard S.

doi:10.1111/j.1432-1033.1997.00619.x

Cited by 23 publications

(11 citation statements)

References 57 publications

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“…The bottom of the pocket, where the p-nitrobenzyl moiety of the ligand is found, has a marked hydrophobic character: seven residues make apolar contacts with the p-nitrobenzyl group. The tight Van der Waals interactions between p-nitrobenzyl and the Fab account for the specificity of D2.3 for pararelative to ortho-nitrosubstituted ligands (10), because the corresponding change in the position of substitution would require a significant rearrangement of the Fab residues to be accommodated.…”

Section: Resultsmentioning

confidence: 99%

“…1); it was obtained by immunizing BALB͞c mice with a protein conjugate of the phosphonate hapten 3 and identified by screening the entire repertoire of hybridomas for catalytic activity (9). D2.3 is the most efficient of the family of antibodies obtained and it accelerates the target reaction by a factor of 10 5 (10). We previously determined the x-ray structure of the Fab D2.3 complexed with TSA 3 at 1.9-Å resolution (8).…”

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confidence: 99%

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X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release

Gigant

Charbonnier

Eshhar

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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The x-ray structures of the unliganded esterase-like catalytic antibody D2.3 and its complexes with a substrate analogue and with one of the reaction products are analyzed. Together with the structure of the phosphonate transition state analogue hapten complex, these crystal structures provide a complete description of the reaction pathway. At alkaline pH, D2.3 acts by preferential stabilization of the negatively charged oxyanion intermediate of the reaction that results from hydroxide attack on the substrate. A tyrosine residue plays a crucial role in catalysis: it activates the ester substrate and, together with an asparagine, it stabilizes the oxyanion intermediate. A canal allows facile diffusion of water molecules to the reaction center that is deeply buried in the structure. Residues bordering this canal provide targets for mutagenesis to introduce a general base in the vicinity of the reaction center.The proposal that antibodies having catalytic activity can be generated to an analogue of a transition state (TSA) of the reaction to be catalyzed (1) has proven to be widely applicable (2, 3). Much has been learned over the last several years about inducing antibodies that promote ester hydrolysis: more than 50 anti-phosphonate antibodies with esterolytic activity have been reported to date. Although extensive steady-state and pre-steady-state kinetic studies have been performed on some antibodies (see ref. 4), the available structural data have focused on the interactions of esterolytic antibodies with phosphonate TSAs (5-8). These confirm the exquisite shape and chemical complementarity between the binding pocket and the haptenic TSA; however, these studies shed little light on the exact pathway that leads substrate to product in an antibody-mediated reaction. Antibody D2.3 catalyses the hydrolysis of the nonactivated p-nitrobenzyl ester 1 (Fig. 1); it was obtained by immunizing BALB͞c mice with a protein conjugate of the phosphonate hapten 3 and identified by screening the entire repertoire of hybridomas for catalytic activity (9). D2.3 is the most efficient of the family of antibodies obtained and it accelerates the target reaction by a factor of 10 5 (10). We previously determined the x-ray structure of the Fab D2.3 complexed with TSA 3 at 1.9-Å resolution (8). We now report the crystal structures of the Fab D2.3 unliganded, complexed with p-nitrobenzyl amide 4 (a substrate analogue inhibitor, SA) and with one of the products of the reaction, p-nitrobenzyl alcohol 2; this allows us to describe key stages along the pathway of the catalyzed reaction. Together with the Fab-TSA 3 structure, the new data support a mechanism whereby hydroxide attacks the carbonyl of the scissile ester bond in the substrate, identify the groups that stabilize the oxyanion intermediate, and define a pathway for the water molecule that gives rise to the attacking hydroxide ion. Only antibody-based haptenprogrammed catalysts, such as D2.3, can be considered for comparing the substrate-catalyst interactions and those betwe...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release

Gigant

Charbonnier

Eshhar

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…In the catalyzed reactions, these residues would be expected to form hydrogen bonds to the oxyanion of the transition state produced by hydroxyl anion or the nucleophilic residue's attack on the substrates, lowering the activation energy. Antibodies D2.3, − D2.4, − D2.5, − and 6D9 − catalyze the hydrolytic reaction by transition-state stabilization. In addition to transition-state stabilization, other catalytic mechanisms, such as nucleophilic and/or general-base catalysis, have been observed or suggested for antibodies 43C9, − 48G7, − CNJ206, − 17E8, − 29G11, and 7C8. − , Although the haptens employed to generate these antibodies do not have a structural entity that induces amino acid residues capable of contributing to nucleophilic and general-base catalysis, antibody diversity has the potential to provide catalytic antibodies possessing these properties in addition to the transition-state stabilization.…”

Section: Hydrolytic Antibodies Generated Against Transition-state Ana...mentioning

confidence: 99%

“…Antibodies D2.3, D2.4, and D2.4 generated with phosphonate 4 catalyzed the hydrolysis of 5 (Scheme , Table ). − These antibodies had rate accelerations that correlated to their affinity for the transition-state analogue relative to the substrate ( K S / K TSA = k cat / k uncat ) (Table ) . The mechanism of the reactions catalyzed by these antibodies is delineated by the transition-state stabilization and by X-ray structural studies .…”

Section: 2 Comparison Of a Set Of Antibodies Generated Against A Sing...mentioning

confidence: 99%

Catalytic Antibodies as Designer Proteases and Esterases

Tanaka

2002

Chem. Rev.

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“…Switching from p-nitrobenzyl to p-nitrophenyl esters served to overcome the reactivity problem and, as discussed above, also reduced product inhibition. § § Tawfik et al (17) subsequently reported p-nitrophenyl ester hydrolytic antibodies elicited by a p-nitrobenzyl phosphonate hapten. In this context, the results reported at about the same time by Jacobsen and Schultz are of interest (18).…”

Section: G3 Catalyzed Cyclizationsmentioning

confidence: 99%

Cyclic peptide formation catalyzed by an antibody ligase

Smithrud¹,

Benkovic²,

Benkovic³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Cyclic hexapeptides represent a class of compounds with important, diverse biological activities. We report herein that the antibody 16G3 catalyzes the cyclization of D-Trp-Gly-Pal-Pro-Gly-Phe⅐p-nitrophenyl ester (8a) to give c-(D-Trp-Gly-Pal-Pro-Gly-L-Phe) (11a). The antibody does not, however, catalyze either epimerization or hydrolysis. The resulting rate enhancement of the cyclization by 16G3 (22-fold) was sufficient to form the desired product in greater than 90% yield. In absolute rate terms, the turnover of 16G3 is estimated to be 2 min ؊1 . The background rate of epimerization of 8a was reduced from 10 to 1% and hydrolysis from 50 to 4% in the presence of 16G3. As expected, the catalytic effects of 16G3 were blocked by the addition of an amount of the hapten equal to twice the antibody concentration. We also synthesized three diaste- S everal years ago, we undertook the generation of antibodies to catalyze peptide bond formation in part because manmade catalysts of peptide bond formation had not been described (1). We were also intrigued by the contrast between medicinal chemistry and antibody design. Generally, the former seeks by design or screening to discover small molecules that interact with macromolecules such as enzymes or receptors leading to enzyme inhibitors or to hormone͞neurotransmitter agonists͞antagonists. In contrast, catalytic antibody research involves the synthesis of haptens, designed to generate novel macromolecules (antibodies), which in turn are capable of catalyzing predetermined chemical reactions. The Design of Hapten 1a: Novel Chemistry and the Generation ofAntibody 16G3 for Bimolecular Peptide Bond Formation. We reported the synthesis of hapten 1a (Fig. 1), designed to induce the formation of antibodies capable of catalyzing the formation of dipeptides of the general structure acetyl-XXX-D-Trp⅐NH 2, wherein XXX represents hydrophobic L-amino acids typified by L-Phe (1). This endeavor generated antibody 16G3, which gave rate enhancements on the order of 2 ϫ 10 4 over the background reaction with pleasingly high turnover rates (Ϸ2 min Ϫ1 ). Our study of the kinetics implicated a sequential mechanism with no preferred order of substrate binding and no evidence for acylation of the antibody by the ester before peptide bond formation. Antibody 16G3 did not, however, catalyze either the hydrolysis or racemization of the active ester. The generation of antibodies that catalyze nonsolvolytic bimolecular bond formation is a greater challenge than the production of antibodies designed to catalyze solvolysis because an important requirement for the former is to prevent hydrolysis. The acylating agents employed in these coupling reactions were the p-nitrophenyl esters of acetyl-XXX. Although the hapten had been designed to generate antibodies that can catalyze the formation only of dipeptides, we subsequently found that antibody 16G3 also catalyzes the coupling of the p-nitrophenyl ester of N-acetyl-LPhe with D-Trp-Gly⅐NH 2 and the p-nitrophenyl ester of N-acetylGly-L-Phe with D-Trp-Gly⅐N...

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Efficient and Selective P‐nitrophenyl‐ester‐hydrolyzing Antibodies Elicited by a P‐nitrobenzyl Phosphonate Hapten

Cited by 23 publications

References 57 publications

X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release

X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release

Catalytic Antibodies as Designer Proteases and Esterases

Cyclic peptide formation catalyzed by an antibody ligase

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