Combining site

By similar logic, protein affinity Hbraries have been constmcted to identify protein—protein combining sites, as in antibody—antigen interaction (19) and recombinant Hbraries have been made which produce a repertoire of antibodies in E. coli (20). In another case, a potential DNA-based therapeutic strategy has been studied (21). DNAs from a partially randomized Hbrary were selected to bind thrombin in vitro. Oligonucleotides, termed aptamers that bound thrombin shared a conserved sequence 14—17 nucleotides long. 

The palytoxin-anti-palytoxin reaction is unique in that its binding increases with increasing temperature (Figure 4). The apparent association constant of the palytoxin to anti-palytoxin was 4.9 x 10 M at 0 C and 1.1 x 10 M" at 35 C, suggesting that H2O must be displaced from some of palytoxin s epitopes before they can bind to their antibody combining sites. 

The antibody-catalyzed Diels-Alder reaction developed by Schultz utilized a Diel-Alderase enzyme-like catalyst evolved from an antibody-combining site (Eq. 12.13). The idea is that the generation of antibodies to a structure that mimics the transition state for the Diels-Alder reaction should result in an antibody-combining site that lowers the entropy of activation by binding both the diene and dienophile in a reactive conformation. 

Figure 4. (a) Selected residues from the combining site of antibody 5C8 com-plexed to piperidine-A/-oxide hapten 4, as determined by X-ray crystallography.1151 The linker portion of the hapten has been truncated to a methyl for comparison with the theozyme complex, (b) The formate/formic acid theozyme complexed to the model of hapten 4, as optimized through quantum mechanical calculations.161... 

Aleshin and coworkers (49) have reported the X-ray crystal structure at 2.2-A resolution of a G2-type variant produced by Aspergillus awamori. Meanwhile, an attempt was made to determine the amino acid residues that participate in the substrate binding and catalysis provided by G2 of A. niger (52). The results of the chemical approach indicated that the Asp-176, Glu-179, and Glu-180 form an acidic cluster crucial to the functioning of the enzyme. This conclusion was then tested by site-specific mutagenesis of these amino acid residues, which were replaced, one at a time, with Asn, Gin, and Gin, respectively (53). The substitution at Glu-179 provided an inactive protein. The other two substitutions affected the kinetic parameters but were not of crucial importance to the maintenance of activity. The crystal structure (49) supports the conclusion that Glu-179 functions as the catalytic acid but Asp-17 6 does not appear to be a good candidate for provision of catalytic base. Thus, there still exists considerable uncertainty as to how the disaccharide is accepted into the combining site for hydrolysis. Nevertheless, the kind of scheme presented by Svensson and coworkers (52) almost surely prevails. 

Competitive immunoassay relies on tne competition between labelled and unlahelled antigens for a fixed and limited number of antibody-combining sites. 

The bait and switch methodology deploys a hapten to act as a bait . This bait is a modified substrate that incorporates ionic functions intended to represent the coulombic distribution expected in the transition state. It is thereby designed to induce complementary, oppositely charged residues in the combining site of antibodies produced by the response of the immune system to this hapten. The catalytic ability of these antibodies is then sought by a subsequent switch to the real substrate and screening for product formation, as described above. 

A similar bait and switch approach has been exploited for acyl-transfer reactions (Janda et al., 1990b, 1991c). The design of hapten [10] incorporates both a transition state mimic and the cationic pyridinium moiety, designed to induce the presence of a potential general acid/base or nucleophilic amino acid residue in the combining site, able to assist in catalysis of the hydrolysis of substrate [11] (Appendix entry 2.6). 

Fig. 10 The use of a cationic hapten [22] mimics the transition state of the base-promoted decomposition of substituted benzisoxazole [20] to cyanophenol [21] and also acts as a bait to induce the presence of an anion in the combining site that...

The bait and switch tactic clearly illustrates that antibodies are capable of a coulombic response that is potentially orthogonal to the use of transition state analogues in engendering catalysis. By variations in the hapten employed, it is possible to fashion antibody combining sites that contain individual residues to deliver intricate mechanisms of catalysis. 

Fig. 38 The mechanism by which an essential Lys residue in the antibody combining site is trapped using the 1,3-diketone [117] to form the covalently linked vinylogous...

This experimental setup was applied for characterization of the interaction between WGA as a targeter and isolated Caco-2 cells, as well as monolayers. Accordingly, all the carbohydrate-combining sites accessible at the cell membrane were occupied within 10 min. This step was followed by internalization of 80% of membrane-bound lectin after 90 min in the case of isolated cells, and 240 min in the case of monolayers [25], The principle of this assay design also works for characterization of the interaction between targeted nanoparticles and monolayers (unpublished data). 

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