Arg-27, Arg-127 and Arg-155 in the β-trefoil protein barley α-amylase/subtilisin inhibitor are interface residues in the complex with barley α-amylase 2

K. W. Rodenburg, E. Varallyay, I. Svendsen, B. Svensson

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Abstract

Arginine residues in barley α-amylase/subtilisin inhibitor (BASI) involved in binding to barley α-amylase 2 (AMY2) were differentially labelled using AMY2 as protectant and phenylglyoxal (PGO) and [14C]PGO as modifying agents, Chymotryptic fragments of labelled BASI were purified by reverse-phase HPLC, and we concluded that the radiolabelled Arg-27, Arg-155 and most likely Arg-127, identified by amino acid, sequence and 14C analyses, are protected by AMY2. While Arg-106 and Arg-107 showed intermediate reactivity and apparently were only partly accessible, Arg-15, Arg-41 and Arg-61 reacted with PGO and were thus exposed in the BASI-AMY2 complex. Patterns of arginine modification by [14C]PGO in free or in AMY2 complexed BASI were consistent with the results of differential labelling. The AMY2-protected arginines in BASI are at a distance from each other, as deduced from crystal structures of different β-trefoil proteins (Erythrina coffra and soybean trypsin inhibitors, interleukin-1α and -1β and WASI, the wheat homologue), suggesting that the BASI-AMY2 complex has multiple contacts at a larger interface. Accordingly, 11-16-residue-long BASI oligopeptides synthesized to include Arg-27, Arg-106/Arg-107 or Arg-127 were unable to suppress the formation of BASI-AMY2 or the effect of an inhibitory monoclonal antibody to BASI: Since Arg-27 is not conserved in rice and wheat AS1s, we further propose that Arg-155 in BASI is the kinetically identified PGO-sensitive group that is essential for inhibition.

Original languageEnglish
Pages (from-to)969-976
Number of pages8
JournalBiochemical Journal
Volume309
Issue number3
DOIs
Publication statusPublished - jan. 1 1995

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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