The Action Pattern of Porcine Pancreatic α-Amylase in Relationship to the Substrate Binding Site of the Enzyme

Abstract

Abstract The position specificity of porcine pancreatic α-amylase action on maltotriose (G3) through maltooctaose (G8) has been studied with maltodextrins specifically labeled in the reducing glucose unit with 14C. The initial relative rates of formation of the labeled products were determined for each substrate. As the size of the maltodextrin increases, the point of maximum frequency of attack shifts from the first bond from the reducing end (bond 1) in G3 to bond 2 in G4 through G7 and to bond 3 in G8. All of the dextrins had more than one bond cleaved except G5, which was cleaved only at bond 2 giving labeled G2 (G2*). Further, neither bond 1 nor the nonreducing terminal bond was cleaved for G5 and higher dextrins in the series. The action patterns on G3 and G4 were observed to be concentration-dependent. At low concentrations of G3* (2.5 mm), G1* was formed at a faster rate than G2*; at 44 mm, the rates of formation of G1* and G2* were equal, and at 100 mm the rate of formation of G2* exceeded G1*. By kinetic analysis and specific labeling experiments, it has been shown that, at low concentrations of G3 the formation of products is due to hydrolysis. At high concentrations, the products are due to a condensation reaction in which 2 molecules of G3 condense to give G6 with subsequent hydrolysis and change in product distribution. The data are consistent with a five-glucose binding site, in which the catalytic groups are located at bond 2.