On porcine pancreatic α-amylase action: kinetic evidence for the binding of two maltooligosaccharide molecules (maltose, maltotriose and o-nitrophenylmaltoside) by inhibition studies: correlation with the five-subsite energy profile

• Published in: European journal of biochemistry Vol. 148; no. 1; pp. 161 - 168
• Main Authors: SEIGNER, C, PRODANOV, E, MARCHIS-MOUREN, G
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 01.04.1985
• Subjects: Allosteric Site; alpha -amylase; alpha-Amylases - antagonists & inhibitors; alpha-Amylases - metabolism; Amylose - metabolism; Analytical, structural and metabolic biochemistry; Animals; Binding, Competitive; Biological and medical sciences; Catalysis; Chemical Phenomena; Chemistry; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glucosides - metabolism; Glucosides - pharmacology; Glycosides - metabolism; Hydrolases; Kinetics; maltose; Maltose - metabolism; Maltose - pharmacology; maltotriose; Models, Chemical; O-nitrophenylmaltoside; Oligosaccharides - metabolism; pancreas; Pancreas - enzymology; pigs; Protein Binding; Substrate Specificity; Swine; Trisaccharides - metabolism; Trisaccharides - pharmacology; Hydrolase; Enzyme; Molecular interaction; Α-Amylase; Pig; Substrate; Vertebrata; Mammalia; Animal; Reaction mechanism; Artiodactyla; Kinetics; Pancreas; Ungulata
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1985.tb08820.x

Hydrolysis of small substrates (maltose, maltotriose and o-nitrophenylmaltoside) catalysed by porcine pancreatic alpha-amylase was studied from a kinetic viewpoint over a wide range of substrate concentrations. Non-linear double-reciprocal plots are obtained at high maltose, maltotriose and o-nitrophenylmaltoside concentrations indicating typical substrate inhibition. These results are consistent with the successive binding of two molecules of substrate per enzyme molecule with dissociation constants Ks1 and Ks2. The Hill plot, log [v/(V-v)] versus log [S], is clearly biphasic and allows the dissociation constants of the ES1 and ES2 complexes to be calculated. Maltose and maltotriose are inhibitors of the amylase-catalysed amylose and o-nitrophenylmaltoside hydrolysis. The inhibition is of the competitive type. The (apparent) inhibition constant Kiapp varies with the inhibitor concentration. These results are also consistent with the successive binding of at least two molecules of maltose or maltotriose per amylase molecule with the dissociation constants Ki1 and Ki2. These inhibition studies show that small substrates and large polymeric ones are hydrolysed at the same catalytic site(s). The values of the dissociation constants Ks1 and Ki1 of the maltose-amylase complexes are identical. According to the five-subsite energy profile previously determined, at low concentration, maltose (as substrate and as inhibitor) binds to the same two sites (4,5) or (3,4), maltotriose (as substrate and as inhibitor) and o-nitrophenyl-maltoside (as substrate) bind to the same three subsites (3,4,5). The dissociation constants Ks2 and Ki2 determined at high substrate and inhibitor concentration are consistent with the binding of the second ligand molecule at a single subsite. The binding mode of the second molecule of maltose (substrate) and o-nitrophenylmaltoside remains uncertain, very likely because of the inaccuracy due to simplifications in the calculations of the subsite binding energies. No binding site(s) outside the catalytic one has been taken into account in this model.