Correlation between Antigen-Combining-Site Structures and Functions within a Panel of Catalytic Antibodies Generated against a Single Transition State Analog

Abstract

The diversity of the immune response, which can provide a panel of catalytic antibodies with varying degrees of catalytic activity and substrate specificity by immunization with a single hapten, raises the question concerning the extent to which a rationally designed hapten dictates the paratopes for catalytic function in the antigencombining site. We have investigated the biochemical properties within a panel of six hydrolytic catalytic antibodies elicited against a phosphonate transition state analog 3 and have examined the correlation between the functions and the antigen-combining-site structures. Although the individual values for kCM, Km, and Ktsa (the affinity for the transition state analog) of the six antibodies differed substantially, the transition state analysis (kcJkuncai versus K$/ A'tsa) displayed a linear relationship (slope = 0.99) with the four antibodies 6D9, 8D11, 4D5, and 9C10, which have homologous primary amino acid sequences, providing evidence that all of the differential binding energy of the transition state vs the ground state is available for the rate enhancement. This also suggested that these four antibodies catalyze the hydrolysis by variations of the same basic mechanism of transition state stabilization. In antibody 6D9, the difference in free energy between the antibody-catalyzed and uncatalyzed reactions was calculated to be 4.0 kcal/mol, a value that is consistent with the typical binding energy for one hydrogen bond between charged and uncharged groups in enzyme-substrate complexes. On the other hand, antibody 7C8, which has an amino acid sequence different from those of the others, deviated from the linear relationship observed in the transition state analysis, suggesting that catalytic factors other than transition state stabilization, such as a functioning acid or base, are involved in the catalysis. Thus, the difference in the catalytic properties is reflected in the differences in the Fv amino acid sequences. The analysis of the substrate specificity suggested that the catalytic antibodies with highly homologous primary amino acid sequences possess homogeneous binding modes to the substrate or hapten. The three-dimensional molecular model of the antibody 6D9-hapten complex reveals that the phosphonate moiety in the hapten interacts with His (L27d), a catalytic amino acid residue participating in the transition state stabilization in the antibody-catalyzed reactions. This histidine is conserved in the catalytic antibodies 6D9, 8D11, 4B5, and 9C10, and chemical modification by DEPC treatment caused a complete loss of the hydrolytic activity. Although the amino acid sequence of antibody 3G6 is highly homologous to the other four catalytic antibodies, antibody 3G6 has Tyr (L27d), which had reduced activity when modified with tetranitromethane. It is noteworthy that the majority of these catalytic antibodies, generated against a single transition state analog, display high homology in the biochemical and structural properties and catalyze the reaction with the same mechanism ...

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.…”

“…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].…”

A free energy calculation study of the effect of H→F substitution on binding affinity in ligand–antibody interactions

“…Antibody 6D9 was generated by immunization with chloramphenicol phosphonate (CP-F) as a hapten, to produce an antibody capable of hydrolyzing a pro-drug of chloramphenicol monoester. [12][13][14][15] Crystal structures of 6D9 containing bound CPD-F have been solved (PDB codes: 1HYX for crystal form I and 1HYY for II). 16 Although crystallization of the antibody in complex with both the original hapten CP-F and its derivative CPD-F was attempted, only the 6D9 -CPD-F complex yielded crystals of high enough quality for determination of the structure at high resolution (1.8 Å); apparently the extra group in CPD-F (compared with CP-F) filled a void in the crystal lattice so as to improve crystal packing.…”

Phage display of a catalytic antibody to optimize affinity for transition-state analog binding

“…Indeed, a number of studies have demonstrated that within a series of related antibodies (9)(10)(11) or enzyme mutants (12), catalytic efficiency usually increases as a function of increased affinity for a transition-state analog. Given the anticipated relationship between activity and hapten binding, we reasoned that in vitro affinity maturation of a catalytic antibody might provide a general means of increasing the catalytic efficiency.…”