title: Covalent reactivity of phosphonate monophenyl esters with serine proteinases: an overlooked feature of presumed transition state analogs.
abstract: Phosphonate monoesters have been assumed to serve as noncovalent transition state analogs for enzymes capable of catalyzing transacylation reactions. Here, we present evidence for the covalent reaction of certain serine proteinases and peptidase antibody fragments with monophenyl amino(4-amidinophenyl)methanephosphonate derivatives. Stable adducts of the N-biotinylated monophenyl ester with trypsin and antibody fragments were evident under conditions that disrupt noncovalent interactions. The reaction was inhibited by the active-site-directed reagent diisopropyl fluorophosphate. Mass spectrometry of the fragments from monoester-labeled trypsin indicated phosphonylation of the active site. Irreversible inhibition of trypsin- and thrombin-catalyzed hydrolysis of model substrates was observed. Kinetic analysis of inactivation of trypsin by the N-benzyloxycarbonylated monoester suggested that the first-order rate constant for formation of covalent monoester adducts is comparable to that of the diester adducts (0.47 vs 2.0 min(-1)). These observations suggest that the covalent reactivity of phosphonate monoesters contributes to their interactions with serine proteinases, including certain proteolytic antibodies.
authors.affiliation: Chemical Immunology and Therapeutics Research Center, Department of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030, USA. yasuhiro.nishiyama@uth.tmc.edu
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Nishiyama
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Taguchi
authors.forename: Jin-Quan
authors.initials: JQ
authors.lastname: Luo
authors.forename: Yong-Xin
authors.initials: YX
authors.lastname: Zhou
authors.forename: Gary
authors.initials: G
authors.lastname: Burr
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Karle
authors.forename: Sudhir
authors.initials: S
authors.lastname: Paul
mesh_terms: D000906:Antibodies / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters; D013058:Mass Spectrometry; D063065:Organophosphonates / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000737:chemistry*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry
chemical_list: D000906:Antibodies; D004952:Esters; D063065:Organophosphonates; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D013917:Thrombin