RBI, a one-domain alpha-amylase/trypsin inhibitor with completely independent binding sites

The bifunctional inhibitor from Ragi (Eleusine coracana Gaertneri) (RBI) is the only member of the alpha-amylase/trypsin inhibitor family that inhibits both trypsin and alpha-amylase. Here, we show that both enzymes simultaneously and independently bind to RBI. The recently solved three-dimensional NMR structure of RBI has revealed that the inhibitor possesses a hitherto unknown fold for serine proteinase and alpha-amylase inhibitors. Despite its different fold, RBI obeys the standard mechanism observed for most protein inhibitors of serine proteinases and is a strong, competitive inhibitor of bovine trypsin (K-i = 1.2 +/- 0.2 nM). RBI is also a competitive inhibitor of porcine alpha-amylase (K-i = 11 +/- 2 nM) when a disaccharide is used as a substrate of alpha-amylase. However, the inhibition mode becomes complex when larger (greater than or equal to 7 saccharide units) alpha-amylase substrates are used. A second saccharide binding site on porcine alpha-amylase may enable larger oligosaccharides to displace RBI from its binding site in an intramolecular reaction.