Substrate-inhibitor interactions in the kinetics of α-amylase inhibition by ragi α-amylase/trypsin inhibitor (RATI) and its various N-terminal fragments

Abstract

The ragi α-amylase/trypsin bifunctional inhibitor (RATI) from Indian finger millet, Ragi (Eleucine coracana Gaertneri), represents a new class of cereal inhibitor family. It exhibits a completely new motif of trypsin inhibitory site and is not found in any known trypsin inhibitor structures. The α-amylase inhibitory site resides at the N-terminal region. These two sites are independent of each other and the inhibitor forms a ternary (1:1:1) complex with trypsin and α-amylase. The trypsin inhibition follows a simple competitive inhibition obeying the canonical serine protease inhibitor mechanism. However, the α-amylase inhibition kinetics is a complex one if larger (≥7 glucose units) substrate is used. While a complete inhibition of trypsin activity can be achieved, the inhibition of amylase is not complete even at very high molar concentration. We have isolated the N-terminal fragment (10 amino acids long) by CNBr hydrolysis of RATI. This fragment shows a simple competitive inhibition of α-amylase activity. We have also synthesized various peptides homologous to the N-terminal sequence of RATI. These peptides also show a normal competitive inhibition of α-amylase with varying potencies. It has also been shown that RATI binds to the larger substrates of α-amylase. In light of these observations, we have reexamined the binding of proteinaceous inhibitors to α-amylase and its implications on the mechanism and kinetics of inhibition.