Dissection of an antibody-catalyzed reaction.

Abstract

Antibody 43C9 accelerates the hydrolysis of a p-nitroanilde by a factor of 2.5 x 10i over the background rate in addition to catalyzing the hydrolysis of a series of aromatic esters. Since this represents one of the largest rate accelerations achieved with an antibody, we have undertaken a series of studies aimed at uncovering the catalytic mechanism of 43C9. The immunogen, a phosphonamidate, was designed to mimic the geometric and electronic characteristics of the tetra- Fig. 1. The data can be fit either to a reaction mechanism involving the titration of a group at the antigenbinding site whose dissociation promotes substrate hydrolysis or to one that features a change in the rate-limiting step around a central antibody-bound intermediate species due to changes in pH. For the p-nitrophenyl ester, the rate constant for p-nitrophenol release (measured independently) approximates ku in the pHindependent region (pH > 9). Thus, the apparent pKa of 9 found for both substrates in the pH rate profiles cannot be due to dissociation of a common acidbase group involved in binding or catalysis. Further support for this conclusion obtains from the absence of pH-dependent binding of 1, a competitive inhibitor ofester hydrolysis that should be sensitive to the ionization state of an active-site acid-base group. The 104 difference in kcw at pH > 9.0 between the two substrates also rules out any mechanism involving a shared conformational change step STo whom reprint requests should be addressed.

Enzyme mimicry by the antiidiotypic antibody approach

“…At the same time, compelling evidence on the covalent catalytic mechanism of 17E8 remains to be obtained (38,39). Covalent catalysis proceeding through an acyl-abzyme intermediate is a feature of another mAb containing HisH35 in the active center, 43C9 (32)(33)(34). Unlike 9A8, comparative mass spectral analysis of mutants and natural 43C9 species showed that the principal nucleophilic residue is HisL91.…”

“…It was shown to participate both in general base and in covalent catalysis (27)(28)(29)(30)(31). In addition, HisH35 is implicated in the stabilization of negative charge in the transition state of esterolysis catalyzed by the 43C9 abzyme and is essential for catalytic activity of this antibody (32)(33)(34). Importantly, mAb 43C9 provides an example of catalysis through a covalent acyl-abzyme intermediate, with another histidine, HisL91, participating as the principal nucleophile.…”

Characterization of the hydrolytic activity of a polyclonal catalytic antibody preparation by pH-dependence and chemical modification studies: evidence for the involvement of Tyr and Arg side chains as hydrogen-bond donors

“…enzyme catalysis and reports of its involvement in antibodymediated catalysis (see, e.g., [7,[9][10][11]41]). Modification of Lys side chains by methyl acetimidate, and of His side chains (up to " 10 per IgG molecule) by DEP resulted in negligible decreases in catalytic activity and binding.…”

“…Chemical modification was targeted at Tyr, Lys and Cys side chains, in view of the kinetically influential ionization observed at pK a " 9, at the side chain of Arg because this has been implicated in the functioning of some other antibody preparations (see, e.g., [7,9,35,41]), and at that of His because of the well known involvement of the imidazole\imidazolium side chain in…”

“…Most investigations have been concerned with monoclonal catalytic antibodies, and a principal objective of the early work was the demonstration of the wide range of chemical reactions for which antibody catalysts could be produced [3,4]. Currently, particular attention is being given to catalytic mechanism [5], and papers reporting kinetic, protein chemical and structural studies are beginning to appear [6][7][8][9][10][11].…”