Anti-hapten 5 mAbs [17E8](MABL-1775)

MABL-1775

Catalytic assays, crystallization, ELISA

Engineer antibody

Species independent

17E 8

Recombinant Antibody

This antibody is specific for norleucine and methionine phenyl esters enantiomers that possess the natural S configuration (L) at the α-carbon.

Noracine phosphonate; Norleucine phenyl phosphonate analog 1; Diphenyl [1-(1-N-Succinylamino)pentyl]phosphonate; Phosphonate transition state analog (hapten)

The original antibody was generated by immunizing Balb/c mice with the KLH conjugate of hapten 5 (norleucine phenyl phosphonate analog 1; Diphenyl [1-(1-N-Succinylamino)pentyl]phosphonate).

This antibody efficiently catalyzes the hydrolysis of N-formyl norleucine phenyl ester and N-formyl methionine phenyl ester, with a preference for N-formyl esters over N-acetyl esters while demonstrating a superior catalytic activity for ester hydrolysis compared to other catalytic antibodies. The catalytic efficiency is influenced by the structural dissimilarity of the substrate to the antigen at specific positions. Notably, the original format of this antibody (mouse IgG2b) exhibits a higher affinity for ester substrates with norleucine side chains than methionine side chains. This original antibody's catalytic activity showed a bell-shaped pH dependence, reaching its maximum at pH 9.5. This pH optimum is attributed to the specific ionizable groups in the antibody's active site, contributing to efficient catalysis. Importantly, this antibody does not display significant product inhibition in kinetics experiments; although weak product inhibition is observed at higher conversion levels, it does not affect the initial rate measurements. Mechanistic studies suggest that this antibody likely forms a potentially rate-limiting covalent acyl intermediate during catalysis, supported by evidence from hydroxylamine partitioning studies (Guo et al., 1994). Crystallization of the Fab-5 complex of this antibody has been accomplished. The structure showed that it has similar active site features to natural triad-based hydrolases. Additionally, the structure showed that hapten five is buried deeply in the antigen-combining site, and there are separate and well-defined binding pockets for the phenyl group and n-butyl side chain of hapten 5 (Zhou et al., 1994; PMID: 8066444) (Wade and Scanlan, 1996).

Guo et al. Kinetic and Mechanistic Characterization of an Efficient Hydrolytic Antibody: Evidence for the Formation of an Acyl Intermediate Journal of the American Chemical Society,116(14), 6062-6069. https://doi.org/10.1021/ja00093a002

The original publication presents the generation and mechanistic characterization of a highly active catalytic antibody that exhibits hydrolytic activity towards norleucine and methionine esters.

100 μg Purified antibody.

1 mg/ml.

Protein A affinity purified

PBS with 0.02% Proclin 300.

Store at 4⁰C for up to 3 months. For longer storage, aliquot and store at - 20⁰C.