A case study NPN43C9-an antibody anilidase

At this point, we can integrate much of what has been discussed above in a single case study. Antibody NPN43C9 was reported in 1988 as the first example of catalysis of hydrolysis of an amide bond, in fact of an active anilide. Its structure and mode of action have been well studied (Janda et al., 1988b), which makes it an appropriate example for this purpose. 

Amide hydrolysis at alkaline pH involves a tetrahedral anionic intermediate, which was mimicked by the transition state analogue [49], an /V-aryl arylphosphonamidate, appropriately related to substrate anilide [50] (Fig. 18) (Appendix entry 2.8). 

The total cDNA construct of NPN43C9 was expressed efficiently in E. coli cells and protein purified, and its catalytic properties were assessed in both the monoclonal antibody and the single-chain antibody (scFv) 7A4-1/212 for the hydrolysis of p-nitroanilide [50] and the related p-chlorophenyl ester [51a] (Fig. 19). Virtually identical kca, and Km values were obtained for both 7A4-1/212 and NPN43C9. This activity was decreased in both cases by the addition of the inhibitor w-nitroanilide [52], which gave K = 800 p.M and 400 pM for the NPN43C9 and 7A4-1/212, respectively. 

NPN43C9 was shown to give a rate acceleration for hydrolysis of [50] of approximately 1.5 X 105, and its values of Km and Fmax were approximately the same as those for its Fab fragment, whose RNA sequence was subsequently used in cloning and expression of Fabs in a bacteriophage A system (Huse et al., 1989). Such an enterprise is capable of giving a greatly 

F g- 20 The proposed catalytic mechanism for hydrolysis of ester substrate [5 If] showing proposed roles for active site residues ArgL9S and HisL91. 

---