Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the ‘flexibility’ hypothesis

Abstract

To investigate the hypothesis that decreased hapten flexibility may lead to increased catalytic antibody activity, we used two closely related immunogens differing only in the flexibility of the atomic framework around the structural motif of the haptens, analogous to the reaction centre of the corresponding substrates. Identical leaving-group determinants in the haptens and identical leaving groups in the substrates removed the ambiguity inherent in some data reported in the literature. Anti-phosphate and anti-phosphonate kinetically homogeneous polyclonal catalytic antibody preparations were compared by using carbonate and ester substrates respectively, each containing a 4-nitrophenolate leaving group. Synthetic routes to a new phosphonate hapten and new ester substrate were developed. The kinetic advantage of the more rigid anti-phosphonate/ester system was demonstrated at pH 8.0 by a 13-fold advantage in k(cat)/k(non-cat) and a 100-fold advantage in the proficiency constant, k(cat)/k (non-cat) x K(m). Despite these differences, the pH-dependences of the kinetic and binding characteristics and the results of chemical modification studies suggest closely similar catalytic mechanisms. The possible origin of the kinetic advantage of the more rigid hapten/substrate system is discussed.

Evidence that the mechanism of antibody-catalysed hydrolysis of arylcarbamates can be determined by the structure of the immunogen used to elicit the catalytic antibody

“…In either event, this result contrasts with that observed for the anti-phosphonamidate antibody used by Wentworth et al [11]. The present study supports the hypothesis of a key role for the presence or absence of the phenolic oxygen in the hapten and contributes to the growing awareness [8,9] that small but key changes in hapten structure can control antibodycatalysed mechanisms.…”

“…One approach to understanding how binding energy can be exploited in catalysis is to investigate how small, relatively subtle, changes in the structures of transition-state analogues affect the kinetic characteristics of the resulting antibodies as catalysts for particular reactions. Some of our recent work aimed at contributing to this objective involved catalytic antibodies elicited by using two closely related immunogens (a phosphate and a phosphonate), differing only in the flexibility of the atomic framework around the structural motifs of the haptens analogous to the reaction centres of the corresponding carbonate ester 1 and carboxylic ester 2 substrates ( Figure 1) [8,9]. The small change in the structure of the hapten, i.e.…”

“…The small change in the structure of the hapten, i.e. removal of one of the phosphate oxygen atoms of 3 in the phosphonate 4 resulted in a catalytic antibody preparation with enhanced catalytic characteristics [8] and increased substrate selectivity [9].…”

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

“…Values for k cat are known to vary to some extent for DNA ligases from other sources (0.02 s −1 for NAD + -dependent DNA ligases from E. coli [1,8]; 0.0025 s −1 for ATP-dependent T4 ligase [40]). These values of k cat are, however, considerably lower than those found for biological catalysts (enzymes [41] and catalytic antibodies generated by active immunization [42]). Also, the fact that k cat and k cat /K m show similar pH profiles over the pH range investigated, the value of K m for S. aureus DNA ligase and AE-labelled DNA substrate used in the present study does not change, remaining at approx.…”

Evidence for ‘lock and key’ character in an anti-phosphonate hydrolytic antibody catalytic site augmented by non-reaction centre recognition: variation in substrate selectivity between an anti-phosphonate antibody, an anti-phosphate antibody and two hydrolytic enzymes

“…Each was generated by one of two closely related immunogens differing only in the flexibility of the atomic framework around the structural motifs of the haptens analogous to the reaction centres of the corresponding (cognate) substrates. As discussed in [12], the systematic study of the relationships between haptenic structure, antibody recognition and catalytic activity is facilitated by the use of polyclonal preparations. Of particular note is that: (i) generation of polyclonal catalytic antibodies samples the entirety of the immune response, (ii) polyclonal IgG preparations investigated to date in different laboratories have not deviated from single-phase (Michaelis-Menten) saturation kinetics, and (iii) polyclonal preparations, at least in our laboratories, are free from contamination by enzymes.…”