alpha-Amylase :: antagonists & inhibitors
[Inhibitors of alpha-amylase from plants--a possibility to treat diabetes mellitus type II by phytotherapy?]
Antidiabetics of plant origin are in common use. A proof of their effectiveness or their mode of action is often missing. The aim of this work was to review the knowledge about inhibitors of alpha-amylase from plants and to comment on the use in anti-diabetic treatment. Herbal alpha-amylase inhibitors are rarely described in the literature, nevertheless they have the ability to lower postprandial blood glucose level and should be used in the supplementary treatment of diabetes. Important constituents for the inhibitory activity against alpha-amylase are mainly polyphenolic compounds. There is a need for further clinical studies to establish a rational therapy with traditional herbal preparations, especially for the leaves from the blueberry, tamarind, lemon balm and rosemary, the hulls from white kidney beans or green tea extract.
Most cited papers:
Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.
Laboratoire d'Architecture et Fonction des Macromolécules Biologiques, UPR9039, Institut de Biologie Structurale et Microbiologie, IFR1, CNRS, Marseille, France.
Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase.
The purification and characterization of an extremely thermostable alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus.
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
The alpha-amylase from Pyrococcus furiosus, a hyperthermophilic archaebacterium, has been purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 66 kDa. The isoelectric point is 4.3. The enzyme displays optimal activity, with substantial thermal stability, at 100 degrees C, with the onset of activity at approximately 40 degrees C. Unlike mesophilic alpha-amylases there is no dependence on Ca2+ for activity or thermostability. The enzyme displays a broad range of substrate specificity, with the capacity to hydrolyze carbohydrates as simple as maltotriose. No subtrate binding occurs below the temperature threshold of activity, and a decrease in Km accompanies an increase in temperature. Except for a decrease in Asp and an increase in Glu, the amino acid composition does not confirm previously defined trends in thermal adaption. Fourth derivative UV spectroscopy and intrinsic fluorescence measurements detected no temperature-dependent structural reorganization. Hydrogen exchange results indicate that the molecule is rigid, with only a slight increase in conformational flexibility at elevated temperature. Scanning microcalorimetry detected no considerable change in the heat capacity function, at the pH of optimal activity, within the temperature range in which activity is induced. The heat absorption peak due to denaturation, under these conditions, occurred within the temperature range of 90-120 degrees C. When the pH was increased, a change in the shape of the heat absorption peak was observed, which when analyzed thermodynamically shows that the process of heat denaturation is complex, and includes at least three stages, indicating that the protein structure consists of three domains. At temperatures below 90 degrees C no excess heat absorption or change in the CD spectra were observed which could be associated with the cooperative conformational transition of the protein. According to the thermodynamic characteristics of the heat denaturation, the cold denaturation of this protein can be expected only at -3 degrees C. Therefore, the observed inactivation of this enzyme is not caused by the cooperative change of its tertiary structure. It can be associated only with the gradual changes of protein domain interaction.
The active center of a mammalian alpha-amylase. Structure of the complex of a pancreatic alpha-amylase with a carbohydrate inhibitor refined to 2.2-A resolution.
LCCMB-CNRS, URA 1296, Faculté de Médecine Nord Bd Pierre Dramard, Marseille, France.
An X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (EC 22.214.171.124) that was soaked with acarbose (a pseudotetrasaccharide alpha-amylase inhibitor) showed electron density corresponding to five fully occupied subsites in the active site. The crystal structure was refined to an R-factor of 15.3%, with a root mean square deviation in bond distances of 0.015 A. The model includes all 496 residues of the enzyme, one calcium ion, one chloride ion, 393 water molecules, and five bound sugar rings. The pseudodisaccharide acarviosine that is the essential structural unit responsible for the activity of all inhibitors of the acarbose type was located at the catalytic center. The carboxylic oxygens of the catalytically competent residues Glu233 and Asp300 form hydrogen bonds with the "glycosidic" NH group of the acarviosine group. The third residue of the catalytic triad Asp197 is located on the opposite side of the inhibitor binding cleft with one of its carbonyl oxygens at a 3.3-A distance from the anomeric carbon C-1 of the inhibitor center. Binding of inhibitor induces structural changes at the active site of the enzyme. A loop region between residues 304 and 309 moves in toward the bound saccharide, the resulting maximal mainchain movement being 5 A for His305. The side chain of residue Asp300 rotates upon inhibitor binding and makes strong van der Waals contacts with the imidazole ring of His299. Four histidine residues (His101, His201, His299, and His305) are found to be hydrogen-bonded with the inhibitor. Many protein-inhibitor hydrogen bond interactions are observed in the complex structure, as is clear hydrophobic stacking of aromatic residues with the inhibitor surface. The chloride activator ion and structural calcium ion are hydrogen-bonded via their ligands and water molecules to the catalytic residues.
Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital Research Institute, Little Rock 72202, USA.
BACKGROUND: Low molecular weight allergens may be responsible for hypersensitivity reactions after the ingestion of wheat. OBJECTIVE: The purpose of this investigation was to identify relevant, low molecular weight allergens after the ingestion of wheat protein. METHODS: Serum samples were collected from seven children with wheat allergy and one adult with baker's asthma. Control serum samples were collected from wheat-tolerant patients. Wheat extracts were prepared and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 12.5% gels revealing numerous protein bands. IgE immunoblot analysis of crude wheat extracts identified multiple IgE-binding proteins. Wheat proteins were separated further with two-dimensional gel electrophoresis, which was followed by IgE immunoblotting investigations. RESULTS: Immunoblot analysis identified a 15 kd wheat protein that bound IgE from all five children with wheat allergy who were evaluated. No IgE binding to this wheat protein was demonstrated in any of the control subjects. Samples representing the 15 kd wheat protein (isoelective point, 5.85) were selected. The N-terminal peptide sequence of this protein (residues 1 to 20) matched to a wheat alpha-amylase inhibitor. CONCLUSION: These data demonstrate that wheat alpha-amylase inhibitor is a relevant allergen in patients experiencing hypersensitivity reactions after the ingestion of wheat protein. This wheat protein, which has been implicated as an important allergen in patients with baker's asthma, represents a sensitizing allergen after both ingestion and inhalation.
Structure of the Aspergillus oryzae alpha-amylase complexed with the inhibitor acarbose at 2.0 A resolution.
Department of Chemistry, University of York, Heslington, York YO1 5DD, England.
The three-dimensional structure of the Aspergillus oryzae alpha-amylase (TAKA-amylase), in complex with the inhibitor acarbose, has been determined by X-ray crystallography at a resolution of 1. 98 A. The tetrasaccharide inhibitor is present as a hexasaccharide presumably resulting from a transglycosylation event. The hexasaccharide occupies the -3 to +3 subsites of the enzyme, consistent with the known number of subsites determined by kinetic studies, with the acarbose unit itself in the -1 to +3 subsites of the enzyme. The transition state mimicking unsaturated pseudo-saccharide occupies the -1 subsite as expected and is present in a distorted 2H3 half-chair conformation. Careful refinement plus extremely well-resolved unbiased electron density suggest that the hexasaccharide represents a genuine transglycosylation product, but the possibility that this apparent species results from an overlapping network of tetrasaccharides is also discussed. Catalysis by alpha-amylase involves the hydrolysis of the alpha-1,4 linkages in amylose with a net retention of the anomeric configuration, via a double-displacement mechanism, as originally outlined by Koshland [Koshland, D. E.(1953) Biol. Rev. 28, 416-336]. The enzymatic acid/base and nucleophile, residues Glu230 and Asp206, respectively, are appropriately positioned for catalysis in this complex, and the hexasaccharide species allows mapping of all the noncovalent interactions between protein and ligand through the enzyme's six subsites.
Determination of the three-dimensional structure of the bifunctional alpha-amylase/trypsin inhibitor from ragi seeds by NMR spectroscopy.
S Strobl, P Mühlhahn, R Bernstein, R Wiltscheck, K Maskos, M Wunderlich, R Huber, R Glockshuber, T A Holak
Max-Planck-Institut für Biochemie, Martinsried, FRG.
The three-dimensional structure of the bifunctional alpha-amylase/trypsin inhibitor (RBI) from seeds of ragi (Eleusine coracana Gaertneri) has been determined in solution using multidimensional 1H and 15N NMR spectroscopy. The inhibitor consists of 122 amino acids, with 5 disulfide bridges, and belongs to the plant alpha-amylase/trypsin inhibitor family for which no three-dimensional structures have yet been available. The structure of the inhibitor was determined on the basis of 1131 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 52 phi angles, supplemented by 9 psi and 51 chi 1 angles. RBI consists of a globular four-helix motif with a simple "up-and-down" topology. The helices are between residues 18-29, 37-51, 58-65, and 87-94. A fragment from Val 67 to Ser 69 and Gln 73 to Glu 75 forms an antiparallel beta-sheet. The fold of RBI represents a new motif among the serine proteinase inhibitors. The trypsin binding loop of RBI adopts the "canonical", substrate-like conformation which is highly conserved among serine proteinase inhibitors. The binding loop is stabilized by the two adjacent alpha-helices 1 and 2. This motif is also novel and not found in known structures of serine proteinase inhibitors. The three-dimensional structure of RBI together with biochemical data suggests the location of the alpha-amylase binding site on the face of the molecule opposite to the site of the trypsin binding loop. The RBI fold should be general for all members of the RBI family because conserved residues among the members of the family from the core of the structure.
Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides.
Department of Biophysics, Institute of Biosciences, Universidade Federal do Rio Grande do Sul, 91.501-970 Porto Alegre, RS, Brazil. email@example.com
To meet the demands for food of the expanding world population, there is need of new ways for protecting plant crops against predators and pathogens while avoiding the use of environmentally aggressive chemicals. A milestone in this field was the introduction into crop plants of genes expressing Bacillus thuringiensis entomotoxic proteins. In spite of the success of this new technology, however, there are difficulties for acceptance of these 'anti-natural' products by the consumers and some concerns about its biosafety in mammals. An alternative could be exploring the plant's own defense mechanisms, by manipulating the expression of their endogenous defense proteins, or introducing an insect control gene derived from another plant. This review deals with the biochemical features and mechanisms of actions of plant proteins supposedly involved in defense mechanisms against insects, including lectins, ribosome-inactivating proteins, enzymes inhibitors, arcelins, chitinases, ureases, and modified storage proteins. The potentialities of genetic engineering of plants with increased resistance to insect predation relying on the repertoire of genes found in plants are also discussed. Several different genes encoding plant entomotoxic proteins have been introduced into crop genomes and many of these insect resistant plants are now being tested in field conditions or awaiting commercialization.
DsbA and DsbC-catalyzed oxidative folding of proteins with complex disulfide bridge patterns in vitro and in vivo.
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland. firstname.lastname@example.org
Oxidative protein folding in the periplasm of Escherichia coli is catalyzed by the thiol-disulfide oxidoreductases DsbA and DsbC. We investigated the catalytic efficiency of these enzymes during folding of proteins with a very complex disulfide pattern in vivo and in vitro, using the Ragi bifunctional inhibitor (RBI) as model substrate. RBI is a 13.1 kDa protein with five overlapping disulfide bonds. We show that reduced RBI can be refolded quantitatively in glutathione redox buffers in vitro and spontaneously adopts the single correct conformation out of 750 possible species with five disulfide bonds. Under oxidizing redox conditions, however, RBI folding is hampered by accumulation of a large number of intermediates with non-native disulfide bonds, while a surprisingly low number of intermediates accumulates under optimal or reducing redox conditions. DsbC catalyzes folding of RBI under all redox conditions in vitro, but is particularly efficient in rearranging buried, non-native disulfide bonds formed under oxidizing conditions. In contrast, the influence of DsbA on the refolding reaction is essentially restricted to reducing redox conditions where disulfide formation is rate limiting. The effects of DsbA and DsbC on folding of RBI in E.coli are very similar to those observed in vitro. Whereas overexpression of DsbA has no effect on the amount of correctly folded RBI, co-expression of DsbC enhanced the efficiency of RBI folding in the periplasm of E.coli about 14-fold. Addition of reduced glutathione to the growth medium together with DsbC overexpression further increased the folding yield of RBI in vivo to 26-fold. This shows that DsbC is the bacterial enzyme of choice for improving the periplasmic folding yields of proteins with very complex disulfide bond patterns.
X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1.7A resolution.
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, U.K.
The three-dimensional structure of the Bacillus stearothermophilus "maltogenic" alpha-amylase, Novamyl, has been determined by X-ray crystallography at a resolution of 1.7 A. Unlike conventional alpha-amylases from glycoside hydrolase family 13, Novamyl exhibits the five-domain structure more usually associated with cyclodextrin glycosyltransferase. Complexes of the enzyme with both maltose and the inhibitor acarbose have been characterized. In the maltose complex, two molecules of maltose are found in the -1 to -2 and +2 to +3 subsites of the active site, with two more on the C and E domains. The C-domain maltose occupies a position identical to one previously observed in the Bacillus circulans CGTase structure [Lawson, C. L., et al.(1994) J. Mol. Biol. 236, 590-600], suggesting that the C-domain plays a genuine biological role in saccharide binding. In the acarbose-maltose complex, the tetrasaccharide inhibitor acarbose is found as an extended hexasaccharide species, bound in the -3 to +3 subsites. The transition state mimicking pseudosaccharide is bound in the -1 subsite of the enzyme in a 2H3 half-chair conformation, as expected. The active site of Novamyl lies in an open gully, fully consistent with its ability to perform internal cleavage via an endo as opposed to an exo activity.
Mechanism of protein stabilization by disulfide bridges: calorimetric unfolding studies on disulfide-deficient mutants of the alpha-amylase inhibitor tendamistat.
Institut für Physikalische, Chemie der Westfälischen, Wilhelms-Universität, Münster, Germany.
The present differential scanning calorimetry and circular dichroism studies on the mechanism of protein stabilization by disulfide bonds were concerned with two questions: is the increase in unfolding entropy upon removal of disulfide links sufficient for the explantation of the general stability decrease of disulfide-deficient mutants? Is it immaterial by which residue cysteine residues are replaced when disulfide bridges are to be opened? To answer these questions we investigated two disulfide bridge mutants of the alpha-amylase inhibitor Tendamistat where the large loop (C45A/C73A) or the small loop (C11A/C27A) had been opened by recombinant DNA techniques, and we compared the stability of the mutated proteins with that of wild-type Tendamistat published previously. To elucidate the significance of the nature of the group that replaces Cys we introduced in position 27 of the small loop four different amino acids instead of Cys: Ala, Leu, Ser and Thr. Surprisingly, opening of the small loop (17 residues) causes larger destabilization than opening of the large loop comprising 29 residues. The thermodynamic parameters at pH 7.0 are: wild-type: t1/2 = 81.6 degrees C, delta Hcal = 296 kJ mol-1, large loop mutant (C45A/C73A): t1/2 = 58.6 degrees C, delta Hcal = 225 kJ mol-1 and small loop mutant (C11A/C27A): t1/2 = 42.7 degrees C, delta Hcal = 135 kJ mol-1. This finding is at variance with the entropy hypothesis. The relative contributions to stability of enthalpic and entropic terms can be varied by a proper choice of substitutions. While the destabilization originating from C45A/C73A exchanges in the large loop turns out to be purely entropic, the stability decreases of the small loop mutants are caused by changes in both enthalpic and entropic terms. Leu or Ser in position 27 leads to an overall enthalpic destabilization. Thr in position 27 increases the transition enthalpy of this mutant to the value of the wild-type protein but increases at the same time the value of the transition entropy with the result of an overall entropic destabilization. Finally, in the C11A/C27A small loop mutant of lowest stability a very large enthalpic destabilization occurs, which is, however, partly counterbalanced by a reduction in the transition entropy. The preferential perturbation of the native state by the mutations is manifest in the increase of the native state heat capacity relative to that of the wild-type protein and the identity of the heat capacity of the unfolded state.(ABSTRACT TRUNCATED AT 400 WORDS)