ja076528m_si_001.pdf (400.22 kB)
Routes to Covalent Catalysis by Reactive Selection for Nascent Protein Nucleophiles
journal contribution
posted on 2007-12-26, 00:00 authored by Andrey V. Reshetnyak, Maria Francesca Armentano, Natalia A. Ponomarenko, Domenica Vizzuso, Oxana M. Durova, Rustam Ziganshin, Marina Serebryakova, Vadim Govorun, Gennady Gololobov, Herbert C. Morse, Alain Friboulet, Sudesh P. Makker, Alexander G. Gabibov, Alfonso TramontanoReactivity-based selection strategies have been used to enrich combinatorial libraries for encoded
biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist
in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites.
The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the
covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied
to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of
chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent
and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the
nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light
chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2.
Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic
site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus
for covalent catalysis.