Selective antibody catalysis

Carrier protein Macromolecule to which a hapten is conjugated, thereby enabling the hapten to stimulate the immune response. catELISA Similar to an ELISA, except that the assay detects catalysis as opposed to simple binding between hapten and antibody. The substrate for a reaction is bound to the surface of the microtitre plate, and putative catalytic antibodies are applied. Any product molecules formed are then detected by the addition of anti-product antibodies, usually in the form of a polyclonal mixture raised in rabbits. The ELISA is then completed in the usual way, with an anti-rabbit second antibody conjugated to an enzyme, and the formation of coloured product upon addition of the substrate for this enzyme. The intensity of this colour is then indicative of the amount of product formed, and thus catalytic antibodies are selected directly. 

Many of the 60 known reactions catalyzed by monoclonal antibodies involve kinetically favored reactions e.g., ester hydrolysis), but abzymes can also speed up kinetically disfavored reactions. Stewart and Benkovic apphed transition-state theory to analyze the scope and limitations of antibody catalysis quantitatively. They found the observed rate accelerations can be predicted from the ratio of equilibrium binding constants of the reaction substrate and the transition-state analogue used to raise the antibody. This approach permitted them to rationalize product selectivity displayed in antibody catalysis of disfavored reactions, to predict the degree of rate acceleration that catalytic antibodies may ultimately afford, and to highlight some differences between the way that they and enzymes catalyze reactions. 

Antibody Catalysis. Recent advances in biocatalysis have led to the generation of catalytic antibodies exhibiting aldolase activity by Lemer and Barbas. The antibody-catalyzed aldol addition reactions display remarkable enantioselectivity and substrate scope [18]. The requisite antibodies were produced through the process of reactive immunization wherein antibodies were raised against a [Tdiketone hapten. During the selection process, the presence of a suitably oriented lysine leads to the condensation of the -amine with the hapten. The formation of enaminone at the active site results in a molecular imprint that leads to the production of antibodies that function as aldol catalysts via a lysine-dependent class I aldolase mechanism (Eq. 8B2.12). 

Many other redox reactions are potentially amenable to antibody catalysis. For example, the chemistry of the P-450 cytochromes, including the hydroxylation of alkanes and the epoxidation of alkenes, can be mimicked with synthetic porphyrins. Incorporation of such molecules into antibody active sites could conceivably yield new catalysts that combine the intrinsic reactivity of the cofactor with the tailored selectivity of the binding pocket. Work is just beginning in this area, but preliminary studies with porphyrin haptens have yielded some interesting results.126-130 Novel redox chemistry can also be anticipated for antibodies containing metal ions, flavins, nicotinamide analogs, and other reactive moieties. 

During the evolution of a natural enzyme, selection is not solely dependent on rate improvement. Therefore, there is no requirement for enzymes to be kinetically perfect, and it should be possible to develop catalytic antibodies that are faster than their natural counterparts. The designed substrate 52 has a rate of 1.4 s-1 with 84G3-catalyzed retro-aldol reaction (Zhong et al., 1999). Its kinetic parameters hold the current world record for antibody catalysis (KM = 4.2 /ulM, = 2 X... 

Further, for antibody catalysis the methyl ester is too small to be an effective epitope but the participation could be induced if an antibody were able to recruit cocaine from the chair conformation to the less stable boat form (see Fig. 4 for the structure) and reorient the syn-protonated amine and benzoyl ester into proximity. Antibodies can provide significant binding energy and in principle antibody binding could effect conformer selection and promotion of substrate-assisted catalysis. To examine this idea, a detailed computational analysis [89] of the energetics of this reaction was also performed for design of novel TSA structures for the alkaline hydrolysis of boat cocaine in comparison with the hydrolysis of chair cocaine. 

Hydrolysis of activated aryl esters, in particular nitrophenyl esters, has been used extensively to demonstrate the principles of antibody catalysis. This type of substrate is advantageous for model studies because formation of the tetrahedral intermediate is rate limiting, so that the issue of guiding decomposition of this intermediate towards hydrolysis does not need to be addressed in design. Nevertheless, hydrolyses of non-activated esters are also catalyzed by antibodies, the most notable example being that of R- and S-selective esterolyses of 2 by antibodies raised against hapten 1 (Scheme 1) [13]. Another anti-1 antibody was shown to promote enantioselective acylation of alcohols such as (S)-4 using vinyl ester 3 as acyl donor [14]. 

Barbas, C.F., Lerner, R.A., and Zhong, G., Antibody catalysis of enantio- and diastereo-selective aldol reactions, Scripps Research Institute, U.S. PatentAppl. US 6210938, 2001 Chem. Abstr., 134, 265244, 2001. 

Traditionally, the desired monoclonal antibody is selected on the basis of binding affinity to the TSA. This approach led to a multitude of effective catalytic antibodies, the rate acceleration of which, however, is usually orders of magnitude below that of comparable enzymes. Furthermore, detailed mechanistic investigations often revealed a different catalysis mechanism than originally assumed. A different approach for the selection of catalytic antibodies is the reactive immunization where the selection criterion from simple binding is changed to chemical reactivity. 

Pollack, 5.J., Jacobs, J.W., Schultz, RG. Selective chemical catalysis by an antibody. Science 234 1570-1573,... 

Janda, K. D., 1997. Chemical selection for catalysis in combinatorial antibody libraries. Science 275 945. 

Conventional ion-selective electrodes have been used as detectors for immunoassays. Antibody binding measurements can be made with hapten-selective electrodes such as the trimethylphenylammonium ion electrode Enzyme immunoassays in which the enzyme label catalyzes the production of a product that is detected by an ion-selective or gas-sensing electrode take advantage of the amplification effect of enzyme catalysis in order to reach lower detection limits. Systems for hepatitis B surface antigen and estradiol use horseradish peroxidase as the enzyme label and... 

Work linking the two classes has been reported by the Kirby and Hilvert groups. The report of Thorn and Hilvert1171 of antibodies that catalysed the Kemp elimination (3 —> 4) remarkable efficiently led Hollfelder et al.1181 to screen a small but carefully selected set of common proteins for evidence of similar catalysis. 

The most interesting developments involve catalysis of simple aldol reactions. The key to reactive immunisation is the use of a hapten that is chemically reactive, rather than a passive template. This means that (i) relevant chemistry is going on during the course of antibody induction, which thus happens in the presence of intermediates involved in the reaction, and so may be modified to favor the formation of antibodies which bind these intermediates (and perhaps transition states leading to them). Furthermore (ii) it becomes possible to select for antibodies that react with, rather than just bind, to the hapten. The system used for the development of aldolase antibodies is outlined in Scheme 2... 

A second use of this type of analysis has been presented by Stewart and Benkovic (1995). They showed that the observed rate accelerations for some 60 antibody-catalysed processes can be predicted from the ratio of equilibrium binding constants to the catalytic antibodies for the reaction substrate, Km, and for the TSA used to raise the antibody, Kt. In particular, this approach supports a rationalization of product selectivity shown by many antibody catalysts for disfavoured reactions (Section 6) and predictions of the extent of rate accelerations that may be ultimately achieved by abzymes. They also used the analysis to highlight some differences between mechanism of catalysis by enzymes and abzymes (Stewart and Benkovic, 1995). It is interesting to note that the data plotted (Fig. 17) show a high degree of scatter with a correlation coefficient for the linear fit of only 0.6 and with a slope of 0.46, very different from the theoretical slope of unity. Perhaps of greatest significance are the... 

Schultz s group employed an a-hydroxyphosphonate hapten [99] and subsequently isolated 20 cell lines of which 5 catalysed the hydrolysis of the model substrate p-nitrophenyl phosphate [100] above background (Fig. 34) (Scanlan et al., 1991). Antibody 38E1 was characterized in more detail and kinetic parameters were afforded by Lineweaver-Burke analysis. This antibody exhibited 11 turnovers per binding site with no change in Vmax, and thus acted as a true catalyst. Moreover, examination of substrate specificity showed that catalysis was entirely selective for p-substituted species (Appendix entry 6.6). 

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