THE MECHANISM OF PORCINE PANCREATIC ALPHA-AMYLASE - KINETIC EVIDENCE FOR 2 ADDITIONAL CARBOHYDRATE-BINDING SITES

Kinetics of inhibition of the two porcine pancreatic alpha-amylase com ponents (PPA I and PPA II) by acarbose were performed using reduced DP 18-maltodextrin and amylose as substrates. Similar Line-weaver-Burk pr imary plots were obtained. Two mixed non-competitive models are propos ed. X-ray crystallographic data [Qian, M., Buisson, G., Duee, E., Hase r, R. & Payan, F. (1991) Biochemistry 33, 6284-6294] are in support of the mixed non-competitive inhibition model which involves abortive co mplexes. Secondary plots are different; inhibition of reduced DP18-mal todextrin hydrolysis gives straight-lines plots while amylose gives pa rabolic curves. These results, confirmed by Dixon-plot analyses, allow us to postulate that, in inhibition of reduced DP18-maltodextrin hydr olysis, one molecule of acarbose is bound/ amylase molecule. In contra st, using amylose as a substrate, two molecules of acarbose are bound. These kinetically determined binding sites might correspond to surfac e sites found by X-ray crystallography [Qian, M., Haser, R. & Payan, F . (1995) Protein Sci. 4, 747-755]; the glucose site close to the activ e site and the maltose site, 2 nm away. In conclusion, no significant difference between PPA I and PPA II has been observed, either from mol ecular mass or from kinetic behaviours; this suggests multiple forms o f the enzyme. A general mechanism of PPA action is proposed; in additi on to the active site, long-chain substrate hydrolysis requires the gl ucose-binding site and the maltose-binding site, while only one site i s necessary for the hydrolysis of short chain substrate.