The mechanism of porcine pancreatic alpha-amylase. Kinetic evidence for two additional carbohydrate-binding sites.
Kinetics of inhibition of the two porcine pancreatic alpha-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, 747-755]; 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.
Alkazaz
M
M
Laboratoire de Biochimie et Biologie de la Nutrition URA-CNRS 1820, Faculté des Sciences et Techniques de St Jérôme, Université d'Aix-Marseille, France.
Desseaux
V
V
Marchis-Mouren
G
G
Payan
F
F
Forest
E
E
Santimone
M
M
D020909:Acarbose; D000688:Amylose / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D010179:Pancreas / Q000201:enzymology*; D011134:Polysaccharides / Q000378:metabolism; D013552:Swine; D014312:Trisaccharides / Q000494:pharmacology; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D011134:Polysaccharides; D014312:Trisaccharides; C019193:maltopentaose; C008315:maltodextrin; D000688:Amylose; D000516:alpha-Amylases; D020909:Acarbose