Studies on the active center of pancreatic amylase

Móra, Susan; Simon, István; Elödi, P

doi:10.1007/bf01731482

Cited by 27 publications

(11 citation statements)

References 13 publications

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“…The binding of three cyclodextrin molecules to α-amylase observed in our crystalline complex is consistent with enzymatic data based on solution studies. Kinetic analyses indicate the binding of three β-cyclodextrin molecules to α-amylase, and, furthermore, show that all three bind with very close to the same affinity, as all have virtually the same dissociation constant (9). Cyclodextrins are not natural substrates of α-amylase.…”

Section: Discussionmentioning

confidence: 99%

“…The “accessory” cyclodextrin (III), which has about the same dissociation constant with the enzyme as the other two cyclodextrins (9), binds to an unusual feature of the protein, a fully solvated and disordered surface tryptophan. We find not only the cyclodextrin at this site, but nearby we have identified a glucose residue associated with Trp203 as was observed by Payan and Qian (30).…”

Section: Discussionmentioning

confidence: 99%

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X-ray Crystallographic Analyses of Pig Pancreatic α-Amylase with Limit Dextrin, Oligosaccharide, and α-Cyclodextrin^,

2010

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Further refinement of the model using maximum likelihood procedures and re-evaluation of the native electron density map has shown that crystals of pig pancreatic α-amylase, whose structure we reported more than fifteen years ago, in fact contain a substantial amount of carbohydrate. The carbohydrate fragments are the products of glycogen digestion carried out as an essential step of the protein's purification procedure. In particular, the substrate-binding cleft contains a limit dextrin of six glucose residues, one of which contains both α-(1,4) and α-(1,6) linkages to contiguous residues. The disaccharide in the original model, shared between two amylase molecules in the crystal lattice, but also occupying a portion of the substrate binding cleft, is now seen to be a tetrasaccharide. There are, in addition, several other probable monosaccharide binding sites. To these results we have further reviewed our X-ray diffraction analysis of α-amylase complexed with α-cyclodextrin. α-Amylase binds three cyclodextrin molecules. Glucose residues of two of the rings superimpose upon the limit dextrin and the tetrasaccharide. The limit dextrin superimposes in large part upon linear oligosaccharide inhibitors visualized by other investigators. By comprehensive integration of these complexes we have constructed a model for the binding of polysaccharides having the helical character known to be present in natural substrates such as starch and glycogen. Keywordsα-amylase; hydrolase; α-cyclodextrin; X-ray crystallography; limit dextrin Pancreatic α-amylase (PPA, α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) was among the first enzymes to be extensively analyzed in terms of substrate binding and catalytic properties (1, 2) and one of the first enzymes to be crystallized (3), yet many of its most salient features remain a mystery. This is due largely to the range of substrates that it will degrade and the diverse structures that those substrates assume. α-Amylase hydrolyzes, without anomeric inversion, α-(1,4) linkages between glucose residues in (1) amylopectin and amylose, which comprise starch, (2) glycogen, (3) oligosaccharides of various lengths, and (4) some cyclodextrins. Both amylopectin and glycogen contain α-(1,6) linkages, which α-amylase † This work was supported by NIH grant GM 080412 to AM. ‡ Atomic coordinates and structure factors for the structures presented here have been deposited in the Protein Data Bank at the Research Collaboratory for Structural Bioinformatics with PDB codes 3L2L and 3L2M.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

X-ray Crystallographic Analyses of Pig Pancreatic α-Amylase with Limit Dextrin, Oligosaccharide, and α-Cyclodextrin^,

2010

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“…The inhibitory effects of cyclodextrins have been studied: the inhibition exerted by β‐cyclodextrin on PPA catalysed amylose hydrolysis is of the competitive type, which shows that β‐cyclodextrin occupies the active center [17]. Also, the inhibitory effects on starch granule hydrolysis exerted by cereal α‐amylases suggested the presence in the enzyme molecule of a noncatalytic binding site [18].…”

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confidence: 99%

Mechanism of porcine pancreatic α‐amylase

Koukiekolo

Desseaux

Moreau

et al. 2001

European Journal of Biochemistry

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The effects of α‐, β‐ and γ‐cyclodextrins on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic α‐amylase (PPA) were investigated. The results of the statistical analysis performed on the kinetic data using the general initial velocity equation of a one‐substrate reaction in the presence of one inhibitor indicate that the type of inhibition involved depends on the substrate used: the inhibition of amylose hydrolysis by α‐, β‐ and γ‐cyclodextrin is of the competitive type, while the inhibition of maltopentaose hydrolysis is of the mixed noncompetitive type. Consistently, the Lineweaver–Burk plots intersect on the vertical axis when amylose is used as the substrate, while in the case of maltopentaose, the intersection occurs at a point located in the second quadrant. The inhibition of the hydrolysis therefore involves only one abortive complex, PPA–cyclodextrin, when amylose is used as the substrate, while two abortive complexes, PPA–cyclodextrin and PPA–maltopentaose–cyclodextrin, are involved with maltopentaose. The mixed noncompetitive inhibition thus shows the existence of one accessory binding site. In any case, only one molecule of inhibitor binds to PPA. In line with these findings, the difference spectra of PPA produced by α‐, β‐ and γ‐cyclodextrin indicate that binding occurs at a tryptophan and a tyrosine residue. The corresponding dissociation constants and the inhibition constants obtained using the kinetic approach are in the same range (1.2–7 mm). The results obtained here on the inhibition of maltopentaose hydrolysis by cyclodextrin are similar to those previously obtained with acarbose as the inhibitor [Alkazaz, M., Desseaux, V., Marchis‐Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100–107], but differ from those obtained with amylose as the substrate and acarbose as inhibitor [Alkazaz, M., Desseaux, V., Marchis‐Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787–796]. It is concluded that the hydrolysis of both long and short chain substrates requires at least one secondary binding site, including a tryptophan residue.

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“…However, almost complete fermentation and hydrolysis by microbial enzymes to form short-chain fatty acids occurs in the large intestine [16,17] . The CDs have been shown to inhibit pancreatic amylase activity [18,19] , and it is therefore plausible that they may inhibit the hydrolysis of complex carbohydrates in the small intestine, leading to a reduced post-prandial glucose response when added to a meal.…”

Section: Introductionmentioning

confidence: 99%

Dose-Dependent Inhibition of the Post-Prandial Glycaemic Response to a Standard Carbohydrate Meal following Incorporation of Alpha-Cyclodextrin

et al. 2006

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Background: This study evaluated the dose-response effects of α-cyclodextrin, a cyclic oligosaccharide, on the glycaemic and insulinaemic responses to the consumption of a standard carbohydrate meal. Methods: In a double-blind, randomised, cross-over design, 10 healthy subjects consumed boiled white rice containing 50 g of digestible carbohydrate to which 0 (control), 2, 5 or 10 g of α-cyclodextrin was added. Plasma glucose and insulin concentrations were determined prior to and for 2 h after consumption of each meal. Results: The area under the plasma glucose curve was negatively related to the dose of α-cyclodextrin (r² = 0.97, p = 0.02), with the areas being significantly reduced at the 5- and 10-gram doses compared with the control (p < 0.05). α-Cyclodextrin did not affect the area under the plasma insulin curve (p = 0.39). Higher doses of α-cyclodextrin resulted in greater satiety, but were associated with reduced palatability and an increased incidence of minor gastrointestinal complaints (stomach ache, nausea, bloating). Conclusion: α-Cyclodextrin reduces the glycaemic response to a standard carbohydrate meal in a dose-dependent manner and may be useful as an ingredient for reducing the glycaemic impact of such foods.

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Studies on the active center of pancreatic amylase

Cited by 27 publications

References 13 publications

X-ray Crystallographic Analyses of Pig Pancreatic α-Amylase with Limit Dextrin, Oligosaccharide, and α-Cyclodextrin^,

X-ray Crystallographic Analyses of Pig Pancreatic α-Amylase with Limit Dextrin, Oligosaccharide, and α-Cyclodextrin^,

Mechanism of porcine pancreatic α‐amylase

Dose-Dependent Inhibition of the Post-Prandial Glycaemic Response to a Standard Carbohydrate Meal following Incorporation of Alpha-Cyclodextrin

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Studies on the active center of pancreatic amylase

Cited by 27 publications

References 13 publications

X-ray Crystallographic Analyses of Pig Pancreatic α-Amylase with Limit Dextrin, Oligosaccharide, and α-Cyclodextrin,

X-ray Crystallographic Analyses of Pig Pancreatic α-Amylase with Limit Dextrin, Oligosaccharide, and α-Cyclodextrin,

Mechanism of porcine pancreatic α‐amylase

Dose-Dependent Inhibition of the Post-Prandial Glycaemic Response to a Standard Carbohydrate Meal following Incorporation of Alpha-Cyclodextrin

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Resources

About

X-ray Crystallographic Analyses of Pig Pancreatic α-Amylase with Limit Dextrin, Oligosaccharide, and α-Cyclodextrin^,

X-ray Crystallographic Analyses of Pig Pancreatic α-Amylase with Limit Dextrin, Oligosaccharide, and α-Cyclodextrin^,