Efficient and selective p-nitrophenyl-ester-hydrolyzing antibodies elicited by a p-nitrobenzyl phosphonate hapten

A number of monoclonal antibodies elicited against a nitrobenzyl (Nbzl)-phosphonate transition-state analogue (TSA), and which were selected for the hydrolysis of the corresponding Nbzl-ester, were also found to catalyze the hydrolysis of the analogous p-nitrophenyl(Np) ester with notable efficiency and specificity. The activity towards the Np-ester is higher in terms of rates (k(cat); as expected from the higher intrinsic reactivity of Np-esters); however, the rate acceleration (k(cat)/k(uncat)) is close to or lower than that observed with the Nbzl-ester. Unexpectedly, the affinity to the Np-ester substrate (1/K(M)) and therefore k(cat)/K(M) are significantly higher. The best example is antibody D2.4 having a k(cat)/K(M) value of 64 s^-1 · M^-1 with the Nbzl-ester and 9400 s^-1 · M^-1 with the Np-ester. Moreover, due to a lower product inhibition by p-nitrophenol relative to p-nitrobenzyl alcohol, these antibodies exhibit more than 1000 turnovers with the Np-ester. The differential affinity of these antibodies to the Nbzl-phosphonate TSA Versus the Nbzl-ester substrate (K(S)/K(TSA) or K(M)/K(i)) correlates well with the observed sate enhancement (k(cat)/k(uncat)). For the Np-ester, however, stabilisation of the transition state (as reflected by K(S)/K(TSA) and by the catalytic proficiencies, k(cat)/K(M)/k(uncat)) does not fully account for the catalytic power (k(cat)/k(uncat)), indicating a more complex catalytic mechanism than simply transition-state stabilization. A comparison of the kinetic parameters of D2.4 with other Np-ester-hydrolyzing antibodies raised against Np-phosphonate haptens emphasizes the marked advantage of this antibody which was elicited against an Nbzl-phosphonate hapten. These results appear to be general: anti-(Nbzl-phosphonate TSA) antibodies obtained from other mouse strains and using different immunization protocols are also efficient Np-esterases. They demonstrate the use of an expanded TSA-hapten, where a spacer (a methylene group) mimics bonds that are partially cleaved in the transition state of the catalyzed reaction.