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 m altopentaose 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 molec ules of inhibitor. In line with this conclusion, the Lineweaver-Burk primar y plots intersect in the second quadrant and the secondary plots of the slo pes and the intercepts versus the inhibitor concentrations are parabolic cu rves, whether the substrate used was amylose or maltopentaose. A specific i nhibition model of the mixed noncompetitive type applies here. This model d iffers 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 pa rticular, with alpha-AI, the inhibition takes place only when PPA and alpha -AI are preincubated together before the substrate is added. This shows tha t the inhibitory PPA-alpha AI complex is formed during the preincubation pe riod. Secondly, other inhibitory complexes are formed, in which two molecul es of inhibitor are bound to either the free enzyme or the enzyme-substrate complex. The catalytic efficiency was determined both with and without inh ibitor. 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.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 mor e 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.