Substrate-inhibitor interactions in the kinetics of alpha-amylase inhibition by ragi alpha-amylase/trypsin inhibitor (RATI) and its various N-terminal fragments.
The ragi alpha-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 alpha-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 alpha-amylase. The trypsin inhibition follows a simple competitive inhibition obeying the canonical serine protease inhibitor mechanism. However, the alpha-amylase inhibition kinetics is a complex one if larger (> or =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 alpha-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 alpha-amylase with varying potencies. It has also been shown that RATI binds to the larger substrates of alpha-amylase. In light of these observations, we have reexamined the binding of proteinaceous inhibitors to alpha-amylase and its implications on the mechanism and kinetics of inhibition.
Alam
N
N
Department of Biophysics, All India Institute of Medical Sciences, New Delhi, 110029, India.
Gourinath
S
S
Dey
S
S
Srinivasan
A
A
Singh
T P
TP
D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D002523:Edible Grain / Q000201:enzymology; D007527:Isoenzymes / Q000138:chemical synthesis / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity; D014361:Trypsin Inhibitors / Q000378:metabolism*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D007527:Isoenzymes; D009842:Oligopeptides; D010446:Peptide Fragments; D010940:Plant Proteins; C406883:RBI protein, Eleusine coracana; D014361:Trypsin Inhibitors; D000516:alpha-Amylases