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

Angelo M. Facchiano, Susan Costantini, Antimo Di Maro, Daniela Panichi, Angela Chambery, Augusto Parente, Simone Di Gennaro, Elia Poerio

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)931-940
Number of pages10
JournalBiological Chemistry
Volume387
Issue number7
DOIs
Publication statusPublished - Jul 1 2006

Keywords

  • MALDI-TOF mass spectrometry
  • Molecular simulations
  • Proteinase inhibitor
  • Proteinase-inhibitor complex
  • Reactive site
  • Recombinant WSCI

ASJC Scopus subject areas

  • Biochemistry

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