Mechanistic study of proton transfer and hysteresis in catalytic antibody 16E7 by site-directed mutagenesis and homology modeling
| Autor: | Jean-Louis Reymond, Ulrich Baumann, Lei Zheng, Wolf Dietrich Woggon, Roman Manetsch |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Stereochemistry
Clinical Biochemistry Pharmaceutical Science Antibodies Catalytic Protonation Biochemistry Immunoglobulin Fab Fragments chemistry.chemical_compound Deprotonation Drug Discovery Homology modeling Cloning Molecular Site-directed mutagenesis Molecular Biology DNA Primers chemistry.chemical_classification Alanine Base Sequence Chemistry Organic Chemistry Benzisoxazole Abzyme Kinetics Models Chemical Mutagenesis Site-Directed Enol ether Molecular Medicine Protons |
| Zdroj: | Bioorganic & Medicinal Chemistry. 13:1021-1029 |
| ISSN: | 0968-0896 |
| DOI: | 10.1016/j.bmc.2004.11.041 |
| Popis: | Antibody 16E7 catalyzes the carbon protonation of enol ether 2 to hemiacetal 3, and the carbon deprotonation of benzisoxazole 7 to phenol 8. This antibody shows an extreme case of hysteresis, requiring several hours to reach full activity. Antibody 16E7 was expressed as recombinant chimeric Fab in Escherichia coli. A model for the three-dimensional structure was produced by homology modeling and used for a docking procedure to obtain models for antibody–ligand complexes. Site-direct mutagenesis of GluL39, identified as a possible catalytic residue by the model, to either glutamine or alanine abolished catalysis, showing that both the protonation reaction of enol ether 2 and the deprotonation of benzisoxazole 7 are promoted by the same residue. The model furthermore suggested that substrate access to the catalytic site might be hindered by a flexible HCDR3 loop held in closed position by a hydrogen bond between SerH99 and GluL39, which could explain the observed hysteresis effect. In agreement with this model, mutagenesis of SerH99 to alanine, or deletion of this residue, was found to reduce hysteresis by approximately 50%. |
| Databáze: | OpenAIRE |
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