Mechanism of porcine pancreatic alpha-amylase inhibition of amylose and maltopentaose hydrolysis by kidney bean (Phaseolus vulgaris) inhibitor and comparison with that by acarbose.
The effects of Phaseolus vulgaris inhibitor (alpha-AI) on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. Based on a statistical analysis of the kinetic data and using the general velocity equation, which is valid at equilibrium for all types of inhibition in a single-substrate reaction, it was concluded that the inhibitory mode is of the mixed noncompetitive type involving two molecules of inhibitor. In line with this conclusion, the Lineweaver-Burk primary plots intersect in the second quadrant and the secondary plots of the slopes and the intercepts versus the inhibitor concentrations are parabolic curves, whether the substrate used was amylose or maltopentaose. A specific inhibition model of the mixed noncompetitive type applies here. This model differs from those previously proposed for acarbose [Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796 and Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107]. In particular, with alpha-AI, the inhibition takes place only when PPA and alpha-AI are preincubated together before the substrate is added. This shows that the inhibitory PPA-alphaAI complex is formed during the preincubation period. Secondly, other inhibitory complexes are formed, in which two molecules of inhibitor are bound to either the free enzyme or the enzyme-substrate complex. The catalytic efficiency was determined both with and without inhibitor. Using the same molar concentration of inhibitor, alpha-AI was found to be a much stronger inhibitor than acarbose. However, when the inhibitor amount is expressed on a weight basis (mg x L-1), the opposite conclusion is drawn. In addition, limited proteolysis was performed on PPA alone and on the alpha-AI-PPA complex. The results show that, in the complex, PPA is more sensitive to subtilisin attack, and shorter fragments are obtained. These data reflect the conformational changes undergone by PPA as the result of the protein inhibitor binding, which differ from those previously observed with acarbose.
Koukiekolo
R
R
Laboratoire de Biochimie et Biologie de la Nutrition CNRS ESA 6033, Faculté des Sciences et techniques de St Jérôme, Université d'Aix-Marseille, France.
Le Berre-Anton
V
V
Desseaux
V
V
Moreau
Y
Y
Rougé
P
P
Marchis-Mouren
G
G
Santimone
M
M
D020909:Acarbose / Q000494:pharmacology; D000688:Amylose / Q000378:metabolism; D000818:Animals; D007887:Fabaceae / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009844:Oligosaccharides / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology*; D010940:Plant Proteins / Q000378:metabolism / Q000494:pharmacology*; D010946:Plants, Medicinal; D011485:Protein Binding; D013552:Swine; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000378:metabolism / Q000494:pharmacology*; D014361:Trypsin Inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D009844:Oligosaccharides; D010940:Plant Proteins; D014361:Trypsin Inhibitors; C019193:maltopentaose; D014359:Trypsin Inhibitor, Kazal Pancreatic; D000688:Amylose; D000516:alpha-Amylases; D020909:Acarbose