Format

Send to

Choose Destination
See comment in PubMed Commons below
J Am Chem Soc. 2007 Dec 26;129(51):16175-82. Epub 2007 Nov 29.

Routes to covalent catalysis by reactive selection for nascent protein nucleophiles.

Author information

1
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10, Miklukho-Maklaya str, Moscow, 117871, Russia.

Abstract

Reactivity-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.

PMID:
18044899
PMCID:
PMC2527816
DOI:
10.1021/ja076528m
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for American Chemical Society Icon for PubMed Central
    Loading ...
    Support Center