Direct Selection for a Catalytic Mechanism from Combinatorial Antibody Libraries

• Published in: Proceedings of the National Academy of Sciences - PNAS 1994-03, Vol.91 (7), p.2532-2536
• Main Authors: Janda, Kim D., Lo, Chih-Hung L., Li, Tingyu, Barbas, Carlos F., Wirsching, Peter
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies; Antibodies, Catalytic - genetics; Antibodies, Catalytic - metabolism; Antibodies, immunoglobulins; Bacteriophages; Biological and medical sciences; Catalysis; Chemicals; Chemistry; Disulfides; Disulfides - metabolism; Enzymes; Esters; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Immunity (Disease); Immunoglobulin Fab Fragments - genetics; Libraries; Medical research; Models, Genetic; Models, Molecular; Molecular immunology; Molecular Sequence Data; Reagents; Selection, Genetic; Structure; Washing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.7.2532

Semisynthetic combinatorial antibody library methodology in the phage-display format was used to select for a cysteine residue in complementarity-determining regions. Libraries were panned with an α-phenethyl pyridyl disulfide that undergoes disulfide interchange. Out of 10 randomly picked clones, two contained an unpaired cysteine, one of which was studied. The antibody catalyzed the hydrolysis of the corresponding thioester where the electrophilic carbonyl occupies the three-dimensional space that was defined by the reactive sulfur atom during selection. The reaction operates by covalent catalysis. Although the steady-state rate enhancement relative to the activated thiol ester substrate is modest, hydrolysis of the acylated cysteine intermediate is remarkably efficient with a catalytic advantage of about four order of magnitude. The results suggest that iterative mechanism-based selection procedures can recapitulate the enzymatic mechanisms refined through evolution.