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Eur J Biochem. 1996 Nov 1;241(3):787-96.

The mechanism of porcine pancreatic alpha-amylase. Kinetic evidence for two additional carbohydrate-binding sites.

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1
Laboratoire de Biochimie et Biologie de la Nutrition URA-CNRS 1820, Faculté des Sciences et Techniques de St Jérôme, Université d'Aix-Marseille, France.

Abstract

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.

PMID:
8944767
[Indexed for MEDLINE]
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