Mechanistically different catalytic antibodies obtained from immunization with a single transition-state analog.

Abstract

The variable-region peptide sequence and steady-state kinetic behavior are compared for a family of catalytic antibodies that arose from the same immune response to a transition-state analog. The crystal structure of the most catalytically active member of the family (17E8) has been solved to 2.5 A resolution and shows that the antibody active site contains a SerH'"-HisH35 (H = heavy chain) catalytic dyad analogous to-the Ser-His-Asp catalytic triad of serine proteases. The variable-region peptide sequence of the next most active antibody (29G11) differs from that of 17E8 by nine heavy-chain point mutations, and results from computer modeling suggest that the three-dimensional structure of 29G11 is similar to that of 17E8. In addition, 29G11 is an efficient catalytic antibody; it possesses 26% of the hydrolytic activity of 17E8. There is one active-site mutation in 29G1l compared to 17E8; position 99 of the heavy chain of 29G11 contains a glycine residue in place of the nucleophilic serine at this position in 17E8. Consistent with this mutation, results from pH-rate studies and hydroxylamine partitioning experiments indicate that in contrast to the catalytic mechanism of 17E8, the mechanism of 29G11-catalyzed esterolysis does not feature nucleophilic catalysis.

Diverse structural solutions to catalysis in a family of antibodies

“…Antibodies resulting from a single immunisation have been defined as belonging to 'a family of antibodies'. A high degree of sequence identity between catalytic antibodies of the same family has been reported in several cases [16][17][18], and when structures were determined, they were found to have many similar features [19,20]. In contrast to these findings, we report here the structure of a catalytic antibody that binds its corresponding TSA hapten in a mode that is totally different from that used by other catalytic antibodies recovered from the same immunisation.…”

Losac, the First Hemolin that Exhibits Procogulant Activity through Selective Factor X Proteolytic Activation

“…In fact, serine proteaselike active sites were ascribed to abzymes: ECL2B-2, i41SL1-2, VIPase (56), and 17E8. In 1994, Zhou et al (61) solved the crystal structure of the complex involving 17E8 and HEP (phenyl[1-(n-succinylamino)pentyl]phosphonate) (Protein Data Bank code 1EAP) (61,62).…”

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].…”