title: Factor Xa active site substrate specificity with substrate phage display and computational molecular modeling.
abstract: Structural origin of substrate-enzyme recognition remains incompletely understood. In the model enzyme system of serine protease, canonical anti-parallel beta-structure substrate-enzyme complex is the predominant hypothesis for the substrate-enzyme interaction at the atomic level. We used factor Xa (fXa), a key serine protease of the coagulation system, as a model enzyme to test the canonical conformation hypothesis. More than 160 fXa-cleavable substrate phage variants were experimentally selected from three designed substrate phage display libraries. These substrate phage variants were sequenced and their specificities to the model enzyme were quantified with quantitative enzyme-linked immunosorbent assay for substrate phage-enzyme reaction kinetics. At least three substrate-enzyme recognition modes emerged from the experimental data as necessary to account for the sequence-dependent specificity of the model enzyme. Computational molecular models were constructed, with both energetics and pharmacophore criteria, for the substrate-enzyme complexes of several of the representative substrate peptide sequences. In contrast to the canonical conformation hypothesis, the binding modes of the substrates to the model enzyme varied according to the substrate peptide sequence, indicating that an ensemble of binding modes underlay the observed specificity of the model serine protease.
authors.affiliation: Genomics Research Center, Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan 115.
authors.forename: Hung-Ju
authors.initials: HJ
authors.lastname: Hsu
authors.forename: Keng-Chang
authors.initials: KC
authors.lastname: Tsai
authors.forename: Yi-Kun
authors.initials: YK
authors.lastname: Sun
authors.forename: Hung-Ju
authors.initials: HJ
authors.lastname: Chang
authors.forename: Yi-Jen
authors.initials: YJ
authors.lastname: Huang
authors.forename: Hui-Ming
authors.initials: HM
authors.lastname: Yu
authors.forename: Chun-Hung
authors.initials: CH
authors.lastname: Lin
authors.forename: Shi-Shan
authors.initials: SS
authors.lastname: Mao
authors.forename: An-Suei
authors.initials: AS
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002021:Buffers; D002417:Cattle; D019295:Computational Biology / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D015951:Factor Xa / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D013379:Substrate Specificity; D017186:Titrimetry
chemical_list: D002021:Buffers; D019151:Peptide Library; D010455:Peptides; D015951:Factor Xa