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

Abstract

The substrate selectivities of an anti-phosphonate and an anti-phosphate kinetically homogeneous polyclonal catalytic antibody preparation and two hydrolytic enzymes were compared by using hapten-analogous and truncated carbonate and ester substrates each containing a 4-nitrophenolate leaving group. Syntheses of the truncated substrates devoid of recognition features in the non-leaving group parts of the substrates are reported. The relatively high kinetic selectivity of the more active anti-phosphonate antibody preparation is considered to depend on a relatively rigid catalytic site with substantial reaction centre specificity together with other important recognition interactions with the extended non-leaving group part of the substrate. In contrast, the less catalytically active, more flexible anti-phosphate antibody exhibits much lower kinetic selectivity for the substrate reaction centre comparable with that of the hydrolytic enzymes with activity much less dependent on recognition interactions with the non-leaving group part of the substrate. The ways in which haptenic flexibility and IgG architecture might contribute to the differential kinetic selectivities are indicated.

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.…”

“…The choice of the particular series of mono-O-aryl N-methyl carbamates 5a-5e was based on (i) the fact that an analogous carbonate, methyl 4-nitrophenyl carbonate 9, which is devoid of the additional recognition features in 1, is a specific substrate for catalytic antibodies generated using the hapten 3 [9], and (ii) the ease of synthesis of suitable 4-substituted phenyl methyl carbamates from commercially available starting materials.…”

“…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].…”

A humanized antibody inhibitor for cathepsin L

“…This protease, therefore, constitutes an attractive target for drug development. However, the generation of small molecule‐ and peptidomimetic‐inhibitors targeting CTSL has proven challenging due to the similar binding pockets and shared catalytic mechanisms among the cathepsin protease family 9 …”