Kinetics of inhibition of the two porcine pancreatic a-amylase components (PPA I and PPA 11) by acarbose were performed using reduced DP18-maltodextrin and amylose as substrates. Similar Lineweaver-Burk primary plots were obtained. Two mixed non-competitive models are proposed. X-ray crystallographic data [Qian, M., Buisson, G., DuCe, E., Haser, R. & Payan, F. (1994) Biochemistry 33, 6284-62941 are in support of the mixed non-competitive inhibition model which involves abortive complexes. Secondary plots are different; inhibition of reduced DPI 8-maltodextrin hydrolysis gives straight-lines plots while amylose gives parabolic curves. These results, confirmed by Dixon-plot analyses, allow us to postulate that, in inhibition of reduced DP18-maltodextrin hydrolysis, one molecule of acarbose is bound amylase molecule. In contrast, using amylose as a substrate, two molecules of acarbose are bound. These kinetically determined binding sites might correspond to surface sites found by X-ray crystallography [Qian, M., Haser, R. & Payan, F. (1995) Protein Sci. 4, 747-7551; the glucose site close to the active site and the maltose site, 2 nm away.In conclusion, no significant difference between PPA I and PPA I1 has been observed, either from molecular mass or from kinetic behaviours; this suggests multiple forms of the enzyme. A general mechanism of PPA action is proposed; in addition to the active site, long-chain substrate hydrolysis requires the glucose-binding site and the maltose-binding site, while only one site is necessary for the hydrolysis of short chain substrate.Keywords: kinetics ; a-amylase; amylose; maltodextrin : acarbose. a-amylases are endoglucanases, catalyzing the hydrolysis of internal a-(1-+4) glucosidic linkages in starch and related polysaccharides. In addition to hydrolysis, they also catalyze condensation and transglycosylation reactions, depending on experimental conditions and especially at high substrate concentrations (Robyt and French, 1970). a-amylases are widely distributed in animals, plants, bacteria and fungi.Two porcine pancreatic components PPA I and PPA I1 have been isolated from tissue homogenate (Marchis-Mouren and Pasero, 1967) on the basis of 1.1 pH differences in isoelectric points (Ajandouz and Marchis-Mouren, 1995). The three-dimensional structure of PPA I (Qian et al., 1993) and PPA I1 (Gilles et al., 1996) at 0.2-nm resolution are identical. Both components and a few other a-amylases, crystallized up to now, belong to the family of barrel enzymes (Farber et al., 1990;Matsuura et al., 1984;Buisson et al., 1987; Boel et al., 1990;Kadziola et al., 1994;Brayer et al., 1995). In addition to the barrel (residues 1 -405), PPA contains a C-terminal domain (residues 406-496) Abbreviations. PPA, porcine pancreatic a-amylase ; DP, degree of polymerisation; r-maltodextrin, reduced DP1 8-maltodextrin; i, vertical axis intercept; El,, pseudo dissociation equilibrium constant; K,,,, apparent Michdelis constant (no inhibitor present).Enzyme. a-(l-4)-glucan-4-glucanohydrolase (EC 3.2.1.1).wit...
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