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 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].
Rodenburg
K W
KW
Carlsberg Laboratory, Department of Chemistry, Copenhagen, Denmark.
Várallyay
E
E
Svendsen
I
I
Svensson
B
B
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D002523:Edible Grain / Q000201:enzymology; D001467:Hordeum / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry / Q000378:metabolism*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D010940:Plant Proteins; C092772:barley alpha-amylase-subtilisin inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean; D001120:Arginine; D000516:alpha-Amylases