ARG-27, ARG-127 AND ARG-155 IN THE BETA-TREFOIL PROTEIN BARLEY ALPHA-AMYLASE SUBTILISIN INHIBITOR ARE INTERFACE RESIDUES IN THE COMPLEX WITH BARLEY ALPHA-AMYLASE-2/

Arginine residues in barley alpha-amylase/subtilisin inhibitor (BASI) involved in binding to barley alpha-amylase 2 (AMY2) were differential ly labelled using AMY2 as protectant and phenylglyoxal (PGO) and [C-14 ]PGO as modifying agents, Chymotryptic fragments of labelled BASI were purified by reverse-phase HPLC, and we concluded that the radiolabell ed Arg-27, Arg-155 and most likely Arg-127, identified by amino acid, sequence and C-14 analyses, are protected by AMY2. While Arg-106 and A rg-107 showed intermediate reactivity and apparently were only partly accessible, Arg-15, Arg-41 and Arg-61 reacted with PGO and were thus e xposed in the BASI-AMY2 complex. Patterns of arginine modification by [C-14]PGO in free or in AMY2-complexed BASI were consistent with the r esults 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 tryp sin inhibitors, interleukin-1 alpha and -1 beta and WASI, the wheat ho mologue), suggesting that the BASI-AMY2 complex has multiple contacts at a larger interface. Accordingly, 11-16-residue-long BASI oligopepti des synthesized to include Arg-27, Arg-106/Arg-107 or Arg-127 were una ble to suppress the formation of BASI-AMY2 or the effect of an inhibit ory 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 kineti cally identified PGO-sensitive group that is essential for inhibition.