Chorismate mutase antibodies

Catalytic antibodies have been obtained for sigmatropic rearrangements, as first demonstrated for the biotransformation of chorismate to prephenate by two chorismate mutase antibodies developed in parallel by Hilvert et al. and Schultz et al. [76]. The Schultz group also described catalysis of an oxy-Cope rearrangement [77]. Schultz et al. and Hilvert et al. have prepared catalytic antibodies for concerted eUminations of aminoxide 53 and selenoxide 54 from immunization... 

Figure 2. Schematic drawing illustrating the hydrogen bonding and electrostatic interactions between the chorismate mutase antibody 1F7 and the transition state analog 4.

Schultz and coworkers (Jackson et a ., 1988) have generated an antibody which exhibits behaviour similar to the enzyme chorismate mutase. The enzyme catalyses the conversion of chorismate [49] to prephenate [50] as part of the shikimate pathway for the biosynthesis of aromatic amino acids in plants and micro-organisms (Haslam, 1974 Dixon and Webb, 1979). It is unusual for an enzyme in that it does not seem to employ acid-base chemistry, nucleophilic or electrophilic catalysis, metal ions, or redox chemistry. Rather, it binds the substrate and forces it into the appropriate conformation for reaction and stabilizes the transition state, without using distinct catalytic groups. 

The conversion of [49] into [50] involves a Claisen rearrangement. Once this was realized it was less surprising that no specific catalytic groups on the enzyme are involved. Support for the Claisen-type mechanism comes from the inhibition shown by the bicyclic dicarboxylate [51], prepared by Bartlett and Johnson (1985) as an analogue of the presumed transition state [52], This same structure [51], coupled through the hydroxyl group to a small protein, was used as a hapten to induce antibodies, one (out of eight) of which mimics the behaviour of chorismate mutase, albeit less efficiently (Table 7). 

Table 7 Chorismate mutase and a catalytic antibody mimic."...

Table 8 Another catalytic antibody mimic of chorismate mutase."...

Using the transition-state analog shown on p. 485 a catalytic antibody with chorismate mutase activity was isolated. Many antibodies catalyzing additional reactions have also been found. Although they are usually less active than natural enzymes, in some cases they approach enzymatic rates. Furthermore, they may catalyze reactions for which no known enzymes exist.h... 

Bowdish, K., Tang, Y, Hicks, J B, and Hilvert, D. (1991) Yeast expression of a catalytic antibody with chorismate mutase activity. J. Biol. Chem 266,11,901—11,908... 

The reaction, which proceeds via a conformationally tight chair-type transition state, is clearly entropically dominated, with a AS of -13 eu. Whereas the known enzyme chorismate mutase from E. coli achieves a 3 x 106-fold accelerated catalysis, the antibody reaches a 104-fold enhancement. A decrease of AS to almost 0 eu points to the presence of an entropy trap. 

Scheme 4.12 Catalytic antibody 1F7 was raised against the transition state analog 28 and possesses modest chorismate mutase activity. It can complement a permissive yeast strain that is auxotrophic for phenylalanine and tyrosine by replacingthe natural enzyme (CM) in the shikimate biosynthetic pathway.

Stereospecificitv. Rate accelerations are only one aspect of enzyme catalyzed reactions. More important for practical applications are the exacting regio- and stereoselectivity displayed by biocatalysts. Since antibodies are chiral molecules, they might be expected to exert considerable control over reactions they promote. In fact, an antibody-catalyzed lactonization reaction was recently reported to be stereospecific (19). Not surprisingly, experiments with racemic chorismate establish that the antibodies with chorismate mutase activity also exhibit high enantioselectivity Q7). 

Fig. 3.6. Chorismate prefers a pseudodiequatorial conformation in solution. It must adopt a disfavored pseudodiaxial conformation to reach the pericyclic transition state. The conformationally constrained oxabicyclic dicarboxylic acid 1, which mimics the transition state, is a potent inhibitor of natural chorismate mutases [62], Antibodies raised against this compound also catalyze the reaction, albeit 100 to 10,000-times less efficiently than their natural counterparts [39, 41].

The Fab fragment of 1F7 has already been shown to function in the cytoplasm of a chorismate mutase-deficient yeast strain [43,44]. When produced at a sufficiently high level, the catalytic antibody is able to replace the missing enzyme and weakly complement the metabolic defect. Conceivably, therefore, it can be placed under selection pressure to identify variants that have higher catalytic efficiency. Preliminary results from such experiments appear quite promising [69]. 

Claisen rearrangement of chorismic acid 1 to prephenic acid 2 (Scheme 1), which is catalyzed by the enzyme chorismate mutase, can be considered as the key step in the biosynthesis of aromatic compounds, that is the so-called shikimic acid pathway. The chair-like transition state geometry 3 was proved by double isotope-labeling experiments [2]. However, in the laboratory this particular reaction can be accelerated not only by enzymes but also by catalytic antibodies [3]. For the generation of such antibodies haptenes such as 4 were used, that is, molecules whose structure is very similar to the transition state of the particular reaction and which are tightly bound by the antibody. 

Fig. 3 A modified Wolfenden plot showing kcat/KM> kcat> and kuncat for a selection of enzymes and antibodies Staphylococcal nuclease (STN), adenosine deaminase (ADA), cytidine deaminase (CDA), alanylalanine transpeptidase (PTE), carboxypeptidase A (CPA), ketosteroid isomerase KSI)y triose phosphate isomerase (TIM), chorismate mutase (CMC), carbonic anhydrase (CAN), cyclophilin (CYC)...

Catalytic antibodies are capable of catalyzing reactions normally carried out by enzymes, albeit usually with much lower efficiency. Interestingly, an x-ray crystallographic analysis of the structure of a catalytic antibody that mimics chorismate mutase showed that it uses essentially the same mechanism to carry out the reaction (45), A similar finding was made for a catalytic antibody with a serine protease active site (46), Both of these observations are fascinating because while enzymes evolved over millions of years, the catalytic antibodies were generated in only a matter of weeks. 

The oxabicyclic inhibitor is making a number of other jpearances as well it has starred in two productions already, and its performance in two more is currently being recorded. The X-ray crystal structures of its complexes with the B. subtilis monofunctional chorismate mutase and with the Hilvert catalytic antibody have been reported by William Lipscomb and Yuh-Min Chook at Harvard, and by Ian Wilson and his coworkers at Scripps, respectively, and similar studies with another enzyme and with the Schultz catalytic antibody are underway. When those studies ate completed, we will have an unprecedented oiqx)ttunity to compare the structures of four different proteins that catalyze the same reaction. 

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