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 (Ki = 1.2 +/- 0.2 nM). RBI is also a competitive inhibitor of porcine alpha-amylase (Ki = 11 +/- 2 nM) when a disaccharide is used as a substrate of alpha-amylase. However, the inhibition mode becomes complex when larger (> or = 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.
Maskos
K
K
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland.
Huber-Wunderlich
M
M
Glockshuber
R
R
D000681:Amylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009844:Oligosaccharides / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D014361:Trypsin Inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D014359:Trypsin Inhibitor, Kazal Pancreatic; D000681:Amylases; D000516:alpha-Amylases; D014357:Trypsin