Primary structure and specificity of the major serine proteinase inhibitor of amaranth ( Amaranthus caudatus L.) seeds

Abstract

A novel of the potato inhibitor I family of serine proteinase inactivating proteins has been isolated from seeds of grain amaranth ( Amaranthus caudatus L.) and characterized. The mature form of the amaranth trypsin/subtilisin inhibitor (ATSI) with p I ≈ 8.3 and molecular mass 7887 Da contains 69 amino acids in a sequence showing 33–51% identity with members of the inhibitor I family from other plant families. A minor form with p I ≈ 7.8 and same inhibitory properties lacked the N-terminal dipeptide Ala-Arg. In accordance with the reactive-site bond Lys 45-Asp 46, which was identified by specific cleavage on a subtilisin column, ATSI is a potent inhibitor of trypsin ( K i ≈ 0.34 nM) and more weakly of plasmin ( K i ≈ 38 nM) and Factor XII a ( K i ≈ 440 nM). However, ATSI also inactivates chymotrypsin ( K i ≈ 0.41 nM), cathepsin G ( K i ≈ 122 nM) and several alkaline microbial proteinases, including subtilisin NOVO ( K i ≈ 0.37 nM). Interestingly, ATSI contains a Trp residue instead of the highly conserved Arg in position 53 (P′ B), which is assumed to play a central role in stabilization of the active-site loop during complex formation. ATSI was immediately inactivated by peptsin and hardly represents an antinutritional component in foods or feeds.