THE INTERACTION OF BARLEY OC-AMYLASE AND BARLEY CC-AMYLASE/SUBTILISIN INHIBITOR AND OF OTHER OC-AMYLASES WITH PROTEINACEOUS INHIBITORS OF CEREAL ORIGIN

摘要

Mature barley seeds contain the proteinaceous oc-amylase/subtilisin inhibitor (BASI) which has been synthesised during grain filling and is a major endosperm protein [1]. BASI specifically inhibits the endogenous barley a-amylase 2 (AMY2), the major isozyme which is de novo synthesised together with isozyme 1 (AMY1) during germination [2]. The role of BASI in vivo may be to inhibit AMY2 to prevent starch mobilisation during premature sprouting and/or to inhibit proteases of the subtilisin family secreted by invading pathogens. BASI belongs to the Kunitz-soybean trypsin inhibitor family [3,4] and other cereals, wheat and rice contain closely related double-headed inhibitors, WASI and RASI [5,6]. The proteins possess a |3-trefoil fold recognised alsoin more distantly related proteins, e.g. interleukins la and 1(3 [7], fibroblast growth factor [8], and hisactophilin [9], but these have no sequence similarity to the trypsin and the double-headed cereal inhibitors [10].The three-dimensional structure of the AMY2/BASI complex has been solved at 1.9 A resolution [11]. It reveals protein-protein interactions that cover a large surface area and a unique fully hydrated calcium ion situated at the protein interface. Thus only indirect contacts through a charged hydrogen bond network containing the hydrated calcium ion connect the catalytic residues in AMY2 with atoms from BASI [11]. Other structures determined of a-amylase/a-amylase inhibitor complexes have either direct contact between catalytic groups and inhibitor atoms, e.g. for tendamistat and porcine pancreatic a-amylase [12], the lectin-like inhibitor (a-AI) and the same enzyme [13], and a Ragi bifunctional inhibitor of the CM-protein family and the closely related yellow meal worm a-amylase [14],or interaction with the catalytic acids occuring via a water molecule as seen in the Amaranth a-amylase inhibitor of the knottin family and the insect a-amylase [15] (Figure 1). Among these systems the lectin-like and the Amaranth inhibitors moreover exert carbohydrate mimicry [13,15]. Only the AMY2/BASI complex consists of proteins from the same organism.