title: Modeling the 3D structure of wheat subtilisin/chymotrypsin inhibitor (WSCI). Probing the reactive site with two susceptible proteinases by time-course analysis and molecular dynamics simulations.
abstract: Comparative modeling and time-course hydrolysis experiments have been applied to investigate two enzyme-inhibitor complexes formed between the wheat subtilisin-chymotrypsin inhibitor (WSCI) and two susceptible proteinases. WSCI represents the first case of a wheat protein inhibitor active against animal chymotrypsins and bacterial subtilisins. The model was created using as template structure that of the CI-2A inhibitor from barley (PDB code: 2CI2), which shares 87% sequence identity with WSCI. Under these conditions of high similarity, the comparative modeling approach can be successfully applied. We predicted the WSCI 3D model and used it to investigate enzyme-inhibitor complex systems. Experimental observations indicated that chymotrypsin, but not subtilisin, in addition to cleavage at the primary reactive site Met48-Glu49, is able to hydrolyze a second peptide bond between Phe58 and Val59. Here, we report on cleavage of the peptide bond at the inhibitor's reactive site (Met48-Glu49) determined using time-course hydrolysis experiments; the same event was investigated for both subtilisin/WSCI and chymotrypsin/WSCI complexes using molecular dynamics simulations. The molecular details of the initial inhibitor-enzyme interactions, as well as of the changes observed during the simulations, allow us to speculate on the different fates of the two WSCI-proteinase complexes.
authors.affiliation: Istituto di Scienze dell'Alimentazione, CNR, I-83100 Avellino, Italy. angelo.facchiano@isa.cnr.it
authors.forename: Angelo M
authors.initials: AM
authors.lastname: Facchiano
authors.forename: Susan
authors.initials: S
authors.lastname: Costantini
authors.forename: Antimo
authors.initials: A
authors.lastname: Di Maro
authors.forename: Daniela
authors.initials: D
authors.lastname: Panichi
authors.forename: Angela
authors.initials: A
authors.lastname: Chambery
authors.forename: Augusto
authors.initials: A
authors.lastname: Parente
authors.forename: Simone
authors.initials: S
authors.lastname: Di Gennaro
authors.forename: Elia
authors.initials: E
authors.lastname: Poerio
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013381:Subtilisins / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; C504099:WSCI protein, Triticum; D013381:Subtilisins