The action pattern of porcine pancreatic alpha-amylase in relationship to the substrate binding site of the enzyme.

The position specificity of porcine pancreatic Lu-amyIase action on maltotriose (Gs) through maltooctaose (Gs) 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 tist bond from the reducing end (bond 1) in G3 to bond 2 in G4 through G7 and to bond 3 in Gs. All of the dextrins had more than one bond cleaved except Gs, which was cleaved only at bond 2 giving labeled Gt (Gz*). Further, neither bond 1 nor the nonreducing terminal bond was cleaved for Ga and higher dextrins in the series. The action patterns on G3 and G4 were observed to be concentration-dependent. At low concentrations of Gs* (2.5 mM), GI* was formed at a faster rate than GZ*; at 44 mM, the rates of formation of G1* and GZ* were equal, and at 100 m&r the rate of formation of Gz* exceeded GI*. By kinetic analysis and specific labeling experiments, it has been shown that, at low concentrations of GO the formation of products is due to hydrolysis. At high concentrations, the products are due to a condensation reaction in which 2 molecules of Ga condense to give Ga 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.