Direct selection for a catalytic mechanism from combinatorial antibody libraries.
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 alpha-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 orders of magnitude. The results suggest that iterative mechanism-based selection procedures can recapitulate the enzymatic mechanisms refined through evolution.
Janda
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KD
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037.
Lo
C H
CH
Li
T
T
Barbas
C F
CF
Wirsching
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P
Lerner
R A
RA
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D004220:Disulfides / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012641:Selection, Genetic
D017151:Antibodies, Catalytic; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments