title: Phosphonate ester probes for proteolytic antibodies.
abstract: The reactivity of phosphonate ester probes with several available proteolytic antibody (Ab) fragments was characterized. Irreversible, active site-directed inhibition of the peptidase activity was evident. Stable phosphonate diester-Ab adducts were resolved by column chromatography and denaturing electrophoresis. Biotinylated phosphonate esters were applied for chemical capture of phage particles displaying Fv and light chain repertoires. Selected Ab fragments displayed enriched catalytic activity inhibitable by the selection reagent. Somewhat unexpectedly, a phosphonate monoester also formed stable adducts with the Abs. Improved catalytic activity of phage Abs selected by monoester binding was evident. Turnover values (kcat) for a selected Fv construct and a light chain against their preferred model peptide substrates were 0.5 and 0.2 min(-1), respectively, and the corresponding Michaelis-Menten constants (Km) were 10 and 8 microm. The covalent reactivity of Abs with phosphonate esters suggests their ability to recapitulate the catalytic mechanism utilized by classical serine proteases.
authors.affiliation: Department of Pathology and Laboratory Medicine, Chemical Immunology and Therapeutics Research Center, University of Texas-Houston Medical School, Houston, Texas 77030, USA. Sudhir.Paul@uth.tmc.edu
authors.forename: S
authors.initials: S
authors.lastname: Paul
authors.forename: A
authors.initials: A
authors.lastname: Tramontano
authors.forename: G
authors.initials: G
authors.lastname: Gololobov
authors.forename: Y X
authors.initials: YX
authors.lastname: Zhou
authors.forename: H
authors.initials: H
authors.lastname: Taguchi
authors.forename: S
authors.initials: S
authors.lastname: Karle
authors.forename: Y
authors.initials: Y
authors.lastname: Nishiyama
authors.forename: S
authors.initials: S
authors.lastname: Planque
authors.forename: S
authors.initials: S
authors.lastname: George
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D001435:Bacteriophages / Q000737:chemistry; D001483:Base Sequence; D002621:Chemistry / Q000379:methods*; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D000906:Antibodies; D004952:Esters; D011506:Proteins; D011993:Recombinant Fusion Proteins