RBI, a one‐domain α‐amylase/trypsin inhibitor with completely independent binding sites

FEBS Letters

Maskos, Klaus¹  Huber-Wunderlich, Martina¹  Glockshuber, Rudi¹ 

[1] Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland

关键词: Ragi bifunctional inhibitor;    Trypsin;    Serine proteinases;    α-Amylase;    Eleusine coracana Gaertneri;    BSA;    bovine serum albumin;    L-BAPA;    N-α-benzoyl-1-arginine-p-nitroanilide;    K i;    inhibition constant;    K M;    Michaelis constant;    NPM;    p-nitrophenyl-α-d-maltoside;    NPMH;    p-nitrophenyl-α-d-maltoheptaoside;    PVDF;    polyvinylidenedifluoride;    Ragi;    Eleusine coracana Gaertneri (Indian finger millet);    RBI;    Ragi bifunctional inhibitor;   

DOI  :  10.1016/S0014-5793(96)01131-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The bifunctional inhibitor from Ragi (Eleusine coracana Gaertneri) (RBI) is the only member of the α-amylase/trypsin inhibitor family that inhibits both trypsin and α-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 α-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 α-amylase (K _i = 11 ± 2 nM) when a disaccharide is used as a substrate of α-amylase. However, the inhibition mode becomes complex when larger (≥7 saccharide units) α-amylase substrates are used. A second saccharide binding site on porcine α-amylase may enable larger oligosaccharides to displace RBI from its binding site in an intramolecular reaction.

【 授权许可】

Unknown