Mechanism of porcine pancreatic α‐amylase

Abstract

The effects of α‐, β‐ and γ‐cyclodextrins on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic α‐amylase (PPA) were investigated. The results of the statistical analysis performed on the kinetic data using the general initial velocity equation of a one‐substrate reaction in the presence of one inhibitor indicate that the type of inhibition involved depends on the substrate used: the inhibition of amylose hydrolysis by α‐, β‐ and γ‐cyclodextrin is of the competitive type, while the inhibition of maltopentaose hydrolysis is of the mixed noncompetitive type. Consistently, the Lineweaver–Burk plots intersect on the vertical axis when amylose is used as the substrate, while in the case of maltopentaose, the intersection occurs at a point located in the second quadrant. The inhibition of the hydrolysis therefore involves only one abortive complex, PPA–cyclodextrin, when amylose is used as the substrate, while two abortive complexes, PPA–cyclodextrin and PPA–maltopentaose–cyclodextrin, are involved with maltopentaose. The mixed noncompetitive inhibition thus shows the existence of one accessory binding site. In any case, only one molecule of inhibitor binds to PPA. In line with these findings, the difference spectra of PPA produced by α‐, β‐ and γ‐cyclodextrin indicate that binding occurs at a tryptophan and a tyrosine residue. The corresponding dissociation constants and the inhibition constants obtained using the kinetic approach are in the same range (1.2–7 mm). The results obtained here on the inhibition of maltopentaose hydrolysis by cyclodextrin are similar to those previously obtained with acarbose as the inhibitor [Alkazaz, M., Desseaux, V., Marchis‐Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100–107], but differ from those obtained with amylose as the substrate and acarbose as inhibitor [Alkazaz, M., Desseaux, V., Marchis‐Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787–796]. It is concluded that the hydrolysis of both long and short chain substrates requires at least one secondary binding site, including a tryptophan residue.

Alpha-Cyclodextrin Attenuates the Glycemic and Insulinemic Impact of White Bread in Healthy Male Volunteers

“…In several in vitro studies it has been shown that alpha-cyclodextrin inhibits the hydrolysis of amylose by porcine pancreatic alpha-amylase, presumably due to the competitive binding of alpha-cyclodextrin to the enzyme's active site [12][13][14][15][16]. The results of the present study can be explained by this mechanism.…”

“…Figure S1: Individual blood glucose levels (mmol/L) versus time (min) after consumption of white bread (red lines) and bread with alpha-CD (blue lines), respectively; Figure S2: Individual blood insulin levels (µIU/mL) versus time (min), after consumption of bread (Treatment A) and bread with alpha-CD (Treatment B), respectively. Figure S3: Time course of individual blood glucose (mmol/L) and blood insulin levels (µIU/mL) sorted by subject number (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and treatment (A: white bread, B: white bread and cyclodextrin, C: alpha-cyclodextrin only).…”

Preparation, physicochemical analysis and molecular modeling investigation of 2,2′-Bipyridine: β-Cyclodextrin inclusion complex in solution and solid state

“…The cyclodextrins also called cycloamyloses, Schardinger dextrins or cycloglycopyranoses are cyclic oligosaccharides in which glucose units are linked by alpha 1-4 glucoside bonds [12,13] The main interests of CDs are their capability to form inclusion complexes with number of compounds [14][15][16][17][18]. X-ray structural analysis reveals that in cyclodextrins the primary hydroxyl groups (C 6 ) are on the narrower edge and the secondary hydroxyl groups (C 2 and C 3 ) are on the wider edge of the ring .…”

The emulsion flocculation stability of protein–carbohydrate diblock copolymers

“…This hypothesis was tested by exposing the adsorbed layers to trypsin or pancreatic α-1,4 amylase digestion. These enzymes were chosen because β-lactoglobulin has trypsin-susceptible bonds [33] and pancreatic α-1,4 amylase digests α-1,4 glycoside links of amylose, amylopectin and related oligosaccharides [34][35][36]. Fig.…”