Kinetic and Mechanistic Characterization of an Efficient Hydrolytic Antibody: Evidence for the Formation of an Acyl Intermediate

Guo, Jincan; Huang, Wei; Scanlan, Thomas S.

doi:10.1021/ja00093a002

Cited by 85 publications

(56 citation statements)

References 2 publications

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“…Heavy chain His35 was found to be a principal catalytic residue in several antibody esterases raised by the TSA approach, as it is likely to be the case in our antibody. It was shown to participate both in general base and in covalent catalysis (27)(28)(29)(30)(31). In addition, HisH35 is implicated in the stabilization of negative charge in the transition state of esterolysis catalyzed by the 43C9 abzyme and is essential for catalytic activity of this antibody (32)(33)(34).…”

Section: Identification Of Catalytic Residues and Modeling Of The 9a8mentioning

confidence: 99%

“…Strikingly, as in the case of 9A8, the 17E8 abzyme also contained SerH99, which potentially completes a catalytic dyad. Based on this observation, we compared the available crystal structure of 17E8 Fab (30,31) with the 9A8 3-D model. Although 17E8 CDRH3 is 3 aa longer than 9A8 CDRH3, superposition of the structures (Fig.…”

Section: Identification Of Catalytic Residues and Modeling Of The 9a8mentioning

confidence: 99%

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Enzyme mimicry by the antiidiotypic antibody approach

Kolesnikov

Kozyr

Alexandrova

et al. 2000

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

110

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The concept of ''internal image'' of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transitionstate analog revealed structural similarity although both antibodies were elicited by two different approaches.

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Section: Identification Of Catalytic Residues and Modeling Of The 9a8mentioning

confidence: 99%

Section: Identification Of Catalytic Residues and Modeling Of The 9a8mentioning

confidence: 99%

Enzyme mimicry by the antiidiotypic antibody approach

Kolesnikov

Kozyr

Alexandrova

et al. 2000

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

110

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“…4). These data for both antibodies can be fit with excellent statistical agreement to a rate equation based on the presence of two ionizable residues in the antibody that mediate catalysis (13). A similar bell-shaped pH-rate profile affording the same pK.…”

Section: Resultsmentioning

confidence: 63%

“…We have reported studies on a catalytic antibody with esterolytic properties (13). The antibody, 17E8, was raised to the norleucine phosphonate hapten 1 and catalyzes the Lenantioselective hydrolysis of formyl norleucine phenyl ester 2 (Fig.…”

mentioning

confidence: 99%

“…The hydroxylamine partitioning behavior of 17E8 and 29G11 was analyzed at the pH corresponding to maximum activity for each antibody (pH 9.5 for 17E8, pH 9.0 for 29G11). This analysis was performed analogously to our previously described method, and the partitioning data for 17E8 (substrate 2, R = acetyl) was obtained in our previous study (13). For the 29G11-catalyzed reaction, the following stock solutions were freshly prepared prior to the experiments: 50 mM ester 2 (R = formyl) in acetonitrile; 29G11 in PBS (3.6 mg/ml); pH 9.0 buffer (50 mM Ches/150 mM NaCl, pH 9.0); 250 mM NH2OH in pH 9.0 buffer; 1.0 M trifluoroacetic acid in water; 10% (wt/vol) FeCl3 in 0.2 M HCI.…”

mentioning

confidence: 99%

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Mechanistically different catalytic antibodies obtained from immunization with a single transition-state analog.

Guo

Huang

Zhou

et al. 1995

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

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The variable-region peptide sequence and steady-state kinetic behavior are compared for a family of catalytic antibodies that arose from the same immune response to a transition-state analog. The crystal structure of the most catalytically active member of the family (17E8) has been solved to 2.5 A resolution and shows that the antibody active site contains a SerH'"-HisH35 (H = heavy chain) catalytic dyad analogous to-the Ser-His-Asp catalytic triad of serine proteases. The variable-region peptide sequence of the next most active antibody (29G11) differs from that of 17E8 by nine heavy-chain point mutations, and results from computer modeling suggest that the three-dimensional structure of 29G11 is similar to that of 17E8. In addition, 29G11 is an efficient catalytic antibody; it possesses 26% of the hydrolytic activity of 17E8. There is one active-site mutation in 29G1l compared to 17E8; position 99 of the heavy chain of 29G11 contains a glycine residue in place of the nucleophilic serine at this position in 17E8. Consistent with this mutation, results from pH-rate studies and hydroxylamine partitioning experiments indicate that in contrast to the catalytic mechanism of 17E8, the mechanism of 29G11-catalyzed esterolysis does not feature nucleophilic catalysis.

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