Arginine residues in barley alpha-amylase/subtilisin inhibitor (BASI) involved in binding to barely alpha-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 beta-trefoil proteins (Erythrina caffra and soybean trypsin inhibitors, interleukin-1 alpha and -1 beta 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 ASIs, we further propose that Arg-155 in BASI is the kinetically identified PGO-sensitive group that is essential for inhibition [Abe, Sidenius and Svensson (1993) Biochem. J. 293, 151-155].
|Publication status||Published - 1995|