Porcine pancreatic α-amylase inhibition by the kidney bean (Phaseolus vulgaris) inhibitor (α-AI1) and structural changes in the α-amylase inhibitor complex

Marius Santimone (1) , Roger Koukiekolo (1) , Yann Moreau (1) , Véronique Le Berre (2, 3) , Pierre Rougé (4) , Guy Marchis-Mouren (1) , Veronique Desseaux (5)

1 IMRN - Institut méditerranéen de Recherche en Nutrition : Biochimie et Biologie de la Nutrition
2 Biochip and Bionanotechnogy
3 LISBP - Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés
4 SCSV - Surfaces Cellulaires et Signalisation chez les Végétaux
5 IMRN - Institut Méditerranéen de Biochimie et Biologie de la Nutrition

Porcine pancreatic a-amylase (PPA) is inhibited by the red kidney bean (Phaseolus vulgaris) inhibitor a-AI1 [Eur. J. Biochem. 265 (1999) 20]. Inhibition kinetics were carried out using DP 4900-amylose and maltopentaose as substrate. As shown by graphical and statistical analysis of the kinetic data, the inhibitory mode is of the mixed noncompetitive type whatever the substrate thus involving the EI, EI2, ESI and ESI2 complexes. This contrast with the E2I complex obtained in the crystal and with biophysical studies. Such difference very likely depends on the [I]/[E] ratio. At low ratio, the E2I complex is favoured; at high ratio the EI, ESI and EI2 complexes are formed. The inhibition model also differs from those previously proposed for acarbose [Eur. J. Biochem. 241 (1996) 787 and Eur. J. Biochem. 252 (1998) 100]. In particular, with a-AI1, the inhibition takes place only when PPA and a-AI are preincubated together before adding the substrate. This indicates that the abortive PPA-aAI1 complex is formed during the preincubation period. One additional carbohydrate binding site is also demonstrated yielding the ESI complex. Also, a second protein binding site is found in EI2 and ESI2 abortive complexes. Conformational changes undergone by PPA upon a-AI1 binding are shown by higher sensitivity to subtilisin attack. From X-ray analysis of the a-AI1-PPA complex (E2I), the major interaction occurs with two hairpin loops L1 (residues 29-46) and L2 (residues 171-189) of a-AI1 protruding into the V-shaped active site of PPA. The hydrolysis of a-AI1 that accounts for the inhibitory activity is reported. D

Mots clés: Enzyme kinetics; a-Amylase; Amylose; Maltopentaose; Phaseolus vulgaris; Inhibition

Domaines: Sciences du Vivant [q-bio]; Biochimie, Biologie Moléculaire; Biologie structurale [q-bio.BM]

Marius Santimone, Roger Koukiekolo, Yann Moreau, Véronique Le Berre, Pierre Rougé, et al.. Porcine pancreatic α-amylase inhibition by the kidney bean (Phaseolus vulgaris) inhibitor (α-AI1) and structural changes in the α-amylase inhibitor complex. Biochimica et Biophysica Acta Proteins and Proteomics, 2004, 1696 (2), pp.181 - 190. ⟨10.1016/j.bbapap.2003.11.001⟩. ⟨hal-01849246⟩