The Mechanism of Porcine Pancreatic α‐Amylase

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Abstract
Kinetics of inhibition of the two porcine pancreatic α‐amylase components (PPA I and PPA II) by acarbose were performed using reduced DP18‐maltodextrin and amylose as substrates. Similar Line‐weaver‐Burk primary plots were obtained. Two mixed non‐competitive models are proposed. X‐ray crystallographic data [ Qian, M., Buisson, G., Duée, E., Haser, R. & Payan, F. (1994) Biochemistry 33, 6284–6294] are in support of the mixed non‐competitive inhibition model which involves abortive complexes. Secondary plots are different; inhibition of reduced DP18‐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 II 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.