Abzymes: Catalytic Antibodies
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By exploiting the highly specific antigen binding properties of antibodies, experimental strategies have been devised to produce antibodies that catalyze chemical reactions. These catalytic antibodies, or abzymes, are selected from monoclonal antibodies generated by immunizing mice with haptens that mimic the transition states of enzyme-catalyzed reactions. For example, the 28B4 abzyme catalyzes periodate oxidation of p-nitrotoluene-methyl sulfide to sulfoxide, as shown below, where electrons from the sulfur atom are transferred to the more electronegative oxygen atom.
The rate of this reaction is promoted by enzyme catalysts that stabilize the transition state of this reaction, thereby decreasing the activation energy and allowing for more rapid conversion of substrate to product. In this case, the transition state is thought to involve a transient positive charge on the sulfur atom and a double-negative charge on the periodate ion as shown below on the left.
In order to generate abzymes complementary in structure to this transition state, mice were immunized with an aminophosphonic acid hapten, as shown above at the right. Obviously, its structure mirrors the structure and electrostatic properties of the sulfoxide transition state. Of the hapten-binding monoclonal antibodies produced with this hapten, many were found to catalyze sulfide oxidation but with a wide range of binding affinities and catalytic efficiencies. In particular, abzyme 28B4 binds hapten with high affinity (Kd = 52 nM) and exhibits a correspondingly high degree of catalytic efficiency (k3/KM = 190,000 M-1s-1).
Elucidation of the molecular structure of abzyme 28B4 bound to the hapten reveals much about the nature of its catalytic action. Highly specific structural and electrostatic interactions create a remarkable degree of structural complementarity between the antigen-binding site and the sulfoxide transition state analog as illustrated in the following series of three-dimensional views of the antibody-hapten complex.
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1. Zoom into the antigen-binding site with bound hapten. Initially, the hapten is visible in the 28B4 Fab fragment bound to the antigen-binding site locted between the H chain and L chain.
2. Zoom in to view the structural complementarity between antibody and hapten.
3. Highlight ionic interactions and hydrogen bonding between sidechain atoms of Arg52, Lys56, & Tyr33 (all in the H chain) and the hapten's anionic phosphate.
4. Highlight hydrophobic and aromatic interactions between several antibody sidechains and the hapten's aromatic group.
5. Highlight p-electron stabilization by the aromatic group of Tyr37 (L chain) and the tetrahedral, cationic nitrogen atom of the hapten.
6. Highlight hydrogen bonding between the sidechain of Asp35 (H chain) and the hapten's electronegative p-nitro (-NO2) group.

Analysis of abzyme 28B4 without bound hapten reveals a slightly altered hapten binding site conformation, suggesting that the induced-fit model of enzyme-substrate binding is a feature of the abzyme's catalytic mechanism. Also, sequence analyses of other antibodies that bind this hapten hint at a mechanism for enzyme evolution. Of particular interest is asparagine 35 of the 28B4 heavy chain which forms a key hydrogen bond with the p-nitro group of the substrate, dramatically increasing the specificity the 28B4 abzyme compared to other hapten-specific antibodies that have a different amino acid residue at this position. The germline sequences of the heavy chain gene segments indicate that this particular asparagine residue arose as the result of somatic mutation of the heavy chain gene during affinity maturation of hapten-specific B lymphocytes. Thus, one can begin to comprehend the evolution of intricately specific enzymes from less-active or inactive protein precursors. The study of catalytic antibodies as a whole has vastly increased current understanding of the mechanisms of enzyme catalysis and represents another step forward in the attempts to create artificially engineered biological enzymes.
Based on the experimental data of L.C.Hsieh-Wilson, P.G.Schultz, and R.C.Stevens. Proc. Natl. Acad. Sci. USA, 93:5363-5367 (1996).

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Sean R. Christensen,
Duane W. Sears
August 27, 2017