Probing the role of the chloride ion in the mechanism of human pancreatic alpha-amylase.

Human pancreatic alpha-amylase (HPA) is a member of the alpha-amylase family involved in the degradation of starch. Some members of this family, including HPA, require chloride for maximal activity. To determine the mechanism of chloride activation, a series of mutants (R195A, R195Q, N298S, R337A, and R337Q) were made in which residues in the chloride ion binding site were replaced. Mutations in this binding site were found to severely affect the ability of HPA to bind chloride ions with no binding detected for the R195 and R337 mutant enzymes. X-ray crystallographic analysis revealed that these mutations did not result in significant structural changes. However, the introduction of these mutations did alter the kinetic properties of the enzyme. Mutations to residue R195 resulted in a 20-450-fold decrease in the activity of the enzyme toward starch and shifted the pH optimum to a more basic pH. Interestingly, replacement of R337 with a nonbasic amino acid resulted in an alpha-amylase that no longer required chloride for catalysis and has a pH profile similar to that of wild-type HPA. In contrast, a mutation at residue N298 resulted in an enzyme that had much lower binding affinity for chloride but still required chloride for maximal activity. We propose that the chloride is required to increase the pK(a) of the acid/base catalyst, E233, which would otherwise be lower due to the presence of R337, a positively charged residue.     

Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase

The mechanism of allosteric activation of alpha-amylase by chloride has been studied through structural and kinetic experiments focusing on the chloride-dependent N298S variant of human pancreatic alpha-amylase (HPA) and a chloride-independent TAKA-amylase. Kinetic analysis of the HPA variant clearly demonstrates the pronounced activating effect of chloride ion binding on reaction rates and its effect on the pH-dependence of catalysis. Structural alterations observed in the N298S variant upon chloride ion binding suggest that the chloride ion plays a variety of roles that serve to promote catalysis. One of these is having a strong influence on the positioning of the acid/base catalyst residue E233. Absence of chloride ion results in multiple conformations for this residue and unexpected enzymatic products. Chloride ion and N298 also appear to stabilize a helical region of polypeptide chain from which projects the flexible substrate binding loop unique to chloride-dependent alpha-amylases. This structural feature also serves to properly orient the catalytically essential residue D300. Comparative analyses show that the chloride-independent alpha-amylases compensate for the absence of bound chloride by substituting a hydrophobic core, altering the manner in which substrate interactions are made and shifting the placement of N298. These evolutionary differences presumably arise in response to alternative operating environments or the advantage gained in a particular product profile. Attempts to engineer chloride-dependence into the chloride-independent TAKA-amylase point out the complexity of this system, and the fact that a multitude of factors play a role in binding chloride ion in the chloride-dependent alpha-amylases.     

A chimera-like alpha-amylase inhibitor suggesting the evolution of Phaseolus vulgaris alpha-amylase inhibitor

White kidney bean (Phaseolus vulgaris) contains two kinds of alpha-amylase inhibitors, one heat-stable (alpha AI-s) and one heat-labile (alpha AI-u). alpha AI-s has recently been revealed to be a tetrameric complex, alpha(2)beta(2), with two active sites [Kasahara et al. (1996) J. Biochem. 120, 177-183]. The present study was undertaken to reveal the molecular features of alpha AI-u, which is composed of three kinds of subunits, alpha, beta, and gamma. The gamma-subunit, in contrast to the alpha- and beta-subunits that are indistinguishable from the alpha- and beta-subunits of alpha AI-s, was found to correspond to a subunit of an alpha-amylase inhibitor-like protein, which has been identified as an inactive, evolutionary intermediate between arcelin and the alpha-amylase inhibitor in a P. vulgaris defense protein family. The polypeptide molecular weight of alpha AI-u determined by the light-scattering technique, together with the polypeptide molecular weights of the subunits, suggests that alpha AI-u is a trimeric complex, alpha beta gamma. The inhibition of alpha AI-u by increasing amounts of porcine pancreatic alpha-amylase (PPA) indicates that an inactive 1:1 complex is formed between alpha AI-u and PPA. Molecular weight estimation of the complex by the light-scattering technique confirmed that it is a complex of alpha AI-u with one PPA molecule. Thus it seems probable that alpha AI-u is an evolutionary intermediate of the P. vulgaris alpha-amylase inhibitor.     

Purification, characterization, and nucleotide sequence of the thermolabile alpha-amylase from the antarctic psychrotroph Alteromonas haloplanctis A23.

The alpha-amylase excreted by the antarctic bacterium Alteromonas haloplanctis was purified and the corresponding amy gene was cloned and sequenced. N- and C-terminal amino acid sequencing were used to establish the primary structure of the mature A. haloplanctis alpha-amylase which is composed of 453 amino acids with a predicted Mr of 49,340 and a pI of 5.5. Three Ca2+ ions are bound per molecule and its activity is modulated by chloride ions. Within the four consensus sequences, Asp-174, Glu-200, and Asp-264 are the proposed catalytic residues. The psychrotrophic A. haloplanctis alpha-amylase is characterized by a high amylolytic activity at low temperatures, a reduced apparent optimal temperature, and typical thermodynamic activation parameters A. haloplanctis alpha-amylase has also a low thermal stability as demonstrated by the temperature effect on both activity and secondary structure. It is suggested that structure flexibility and lower sensitivity of secondary structure to temperature variations in the low temperature range are the main structural adaptations of the psychrotrophic enzyme. The unusual stacking of small amino acids around the catalytic residues is proposed as a factor inducing active site flexibility and concomitant high activity of the enzyme at low temperatures.     

Inhibitory effect of 0.19 alpha-amylase inhibitor from wheat kernel on the activity of porcine pancreas alpha-amylase and its thermal stability

The inhibitory effect of 0.19 alpha-amylase inhibitor (0.19 AI) from wheat kernel on the porcine pancreas alpha-amylase (PPA)-catalyzed hydrolysis of p-nitrophenyl-alpha-D-maltoside (pNP-G2) was examined. 0.19 AI is a homodimer of 26.6 kDa with 13.3-kDa subunits under the conditions used. The elution behaviors in gel filtration HPLC of PPA and 0.19 AI indicated that a PPA molecule bound with a 0.19 AI molecule (homodimer) at a molar ratio of 1:1. 0.19 AI inhibited PPA activity in a competitive manner with an inhibitor constant, K(i), of 57.3 nM at pH 6.9, 30 degrees C, and the binding between them was found to be endothermic and entropy-driven. The activation energy for the thermal inactivation of 0.19 AI was determined to be 87.0 kJ/mol, and the temperature, T(50), giving 50% inactivation in a 30-min incubation at pH 6.9 was 88.1 degrees C. The high inhibitory activity of 0.19 AI against PPA and its high thermal stability suggest its potential for use in the prevention and therapy of obesity and diabetes.     

Probing the catalytically essential residues of the alpha-L-fucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus

Retaining glycosidases promote the hydrolysis of the substrate by following a double-displacement mechanism involving a covalent intermediate. The catalytic residues are a general acid/base catalyst and the nucleophile. Experimental identification of these residues in a specific glycosidase allows for the assigning of the corresponding residues in all of the other enzymes belonging to the same family. By means of sequence alignment, mutagenesis, and detailed kinetic studies of the alpha-fucosidase from Sulfolobus solfataricus (Ssalpha-fuc) (family 29), we show here that the residues, invariant in this family, have the function inferred from the analysis of the 3D structure of the enzyme from Thermotoga maritima (Tmalpha-fuc). These include in Ssalpha-fuc the substrate-binding residues His46 and His123 and the nucleophile of the reaction, previously described. The acid/base catalyst could be assigned less easily. The k(cat) of the Ssalpha-fucGlu292Gly mutant, corresponding to the acid/base catalyst of Tmalpha-fuc, is reduced by 154-fold but could not be chemically rescued. Instead, the Ssalpha-fucGlu58Gly mutant revealed a 4000-fold reduction of k(cat)/K(M) if compared to the wild-type and showed the rescue of the k(cat) by sodium azide at wild-type levels. Thus, our data suggest that a catalytic triad, namely, Glu58, Glu292, and Asp242, is involved in catalysis. Glu58 and Glu292 cooperate in the role of acid/base catalyst, while Asp242 is the nucleophile of the reaction. Our data suggest that in glycosidase family 29 alpha-fucosidases promoting the retaining mechanism with slightly different catalytic machineries coexist.     

Identification of key amino acid residues in Neisseria polysaccharea amylosucrase

Amylosucrase from Neisseria polysaccharea catalyzes the synthesis of an amylose-like polymer from sucrose. Sequence alignment revealed that it belongs to the glycoside hydrolase family 13. Site-directed mutagenesis enabled the identification of functionally important amino acid residues located at the active center. Asp-294 is proposed to act as the catalytic nucleophile and Glu-336 as general acid base catalyst in amylosucrase. The conserved Asp-401, His-195 and His-400 residues are critical for the enzymatic activity. These results provide strong support for the predicted close structural and functional relationship between the sucrose-glucosyltransferases and enzymes of the alpha-amylase family.     

Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.

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.     

Alternative catalytic anions differentially modulate human alpha-amylase activity and specificity

A mechanistic study of the essential allosteric activation of human pancreatic alpha-amylase by chloride ion has been conducted by exploring a wide range of anion substitutions through kinetic and structural experiments. Surprisingly, kinetic studies indicate that the majority of these alternative anions can induce some level of enzymatic activity despite very different atomic geometries, sizes, and polyatomic natures. These data and subsequent structural studies attest to the remarkable plasticity of the chloride binding site, even though earlier structural studies of wild-type human pancreatic alpha-amylase suggested this site would likely be restricted to chloride binding. Notably, no apparent relationship is observed between anion binding affinity and relative activity, emphasizing the complexity of the relationship between chloride binding parameters and the activation mechanism that facilitates catalysis. Of the anions studied, particularly intriguing in terms of observed trends in substrate kinetics and their novel atomic compositions were the nitrite, nitrate, and azide anions, the latter of which was found to enhance the relative activity of human pancreatic alpha-amylase by nearly 5-fold. Structural studies have provided considerable insight into the nature of the interactions formed in the chloride binding site by the nitrite and nitrate anions. To probe the role such interactions play in allosteric activation, further structural analyses were conducted in the presence of acarbose, which served as a sensitive reporter molecule of the catalytic ability of these modified enzymes to carry out its expected rearrangement by human pancreatic alpha-amylase. These studies show that the largest anion of this group, nitrate, can comfortably fit in the chloride binding pocket, making all the necessary hydrogen bonds. Further, this anion has nearly the same ability to activate human pancreatic alpha-amylase and leads to the production of the same acarbose product. In contrast, while nitrite considerably boosts the relative activity of human pancreatic alpha-amylase, its presence leads to changes in the electrostatic environment and active site conformations that substantially modify catalytic parameters and produce a novel acarbose rearrangement product. In particular, nitrite-substituted human pancreatic alpha-amylase demonstrates the unique ability to cleave acarbose into its acarviosine and maltose parts and carry out a previously unseen product elongation. In a completely unexpected turn of events, structural studies show that in azide-bound human pancreatic alpha-amylase, the normally resident chloride ion is retained in its binding site and an azide anion is found bound in an embedded side pocket in the substrate binding cleft. These results clearly indicate that azide enzymatic activation occurs via a mechanism distinct from that of the nitrite and nitrate anions.     

Continuing efforts on the improvement of Beckmann rearrangement of indanone oxime

Continuing search for beneficial additive toward the Beckmann rearrangement (BR) of indanone oxime has revealed that common Lewis acid catalyst in methanesulfonyl chloride (MsCl) showed increasing efficiency in this ionic rearrangement. The new protocol with MsCl is superior to the classical phosphorus-based methods such as PPA and Eaton reagent, especially in the reaction of indanone oximes.     

Knowledge-based model of a glucosyltransferase from the oral bacterial group of mutans streptococci.

Mutans streptococci glucosyltransferases catalyze glucosyl transfer from sucrose to a glucan chain. We previously identified an aspartyl residue that participates in stabilizing the glucosyl transition state. The sequence surrounding the aspartate was found to have substantial sequence similarity with members of alpha-amylase family. Because little is known of the protein structure beyond the amino acid sequence, we used a knowledge-based interactive algorithm, MACAW, which provided significant level of homology with alpha-amylases and glucosyltransferase from Streptococcus downei gtfI (GTF). The significance of GTF similarity is underlined by GTF/alpha-amylase residues conserved in all but one alpha-amylase invariant residues. Site-directed mutagenesis of the three GTF catalytic residues are homologous with the alpha-amylase catalytic triad. The glucosyltransferases are members of the 4/7-superfamily that have a (beta/alpha)8-barrel structure and belong to family 13 of the glycohydralases.     

Carbohydrate binding sites in a pancreatic alpha-amylase-substrate complex, derived from X-ray structure analysis at 2.1 A resolution.

The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) that was soaked with the substrate maltopentaose showed electron density corresponding to two independent carbohydrate recognition sites on the surface of the molecule. Both binding sites are distinct from the active site described in detail in our previous high-resolution study of a complex between PPA and a carbohydrate inhibitor (Qian M, Buisson G, Duée E, Haser H, Payan F, 1994, Biochemistry 33:6284-6294). One of the binding sites previously identified in a 5-A-resolution electron density map, lies at a distance of 20 A from the active site cleft and can accommodate two glucose units. The second affinity site for sugar units is located close to the calcium binding site. The crystal structure of the maltopentaose complex was refined at 2.1 A resolution, to an R-factor of 17.5%, with an RMS deviation in bond distances of 0.007 A. The model includes all 496 residues of the enzyme, 1 calcium ion, 1 chloride ion, 425 water molecules, and 3 bound sugar rings. The binding sites are characterized and described in detail. The present complex structure provides the evidence of an increased stability of the structure upon interaction with the substrate and allows identification of an N-terminal pyrrolidonecarboxylic acid in PPA.     

Isozymes of alpha-amylase in the porcine pancreas: population distribution

1. Three isozymes of pancreatic alpha-amylase, PPA 1, PPA 2, and PPA 3, were observed in a porcine population of 50 animals. 2. Isozyme PPA 2 was common to each pancreas. 3. Three phenotypic patterns were described as: (A) consisting of PPA 2 alone (20%); (B) consisting of PPA 1 and PPA 2 (78%); and (C) consisting of all three forms (2%). 4. Amylase isozymes were separated by anion exchange chromatography using DE53. 5. Individual isozymes corresponded to one of the three isozymes found in pancreatin. 6. Individual isozymes were inhibited equally by an amylase inhibitor from wheat. 7. Differences in amylase isozymes were attributed to genetically controlled mechanisms and not to artifacts of isolation.     

Characterization of alpha-amylase inhibitor from Palo Fierro seeds.

Alpha amylase inhibitor from Palo Fierro seeds (alphaAI-PF) was purified using affinity chromatography on a fetuin-fractogel column followed by anionic exchange chromatography. AlphaAI-PF has a molecular mass of 77kDa with two subunits (15.8 and 17.4 kDa), it is nonglycosylated and has pI of 4.7. AlphaAI-PF inhibited porcine pancreatic alpha-amylase (PPA) (1,4-alpha-D-glucan glucanohydrolase; EC 3.2.1.1), but was almost devoid of inhibitory activity on alpha-amylase extracts from Zabrotes subfasciatus (ZSA). Analysis of alphaAI-PF peptides showed a high homology to alphaAI-1 from Phaseolus vulgaris that also inhibits PPA.     

New substrate specificity of modified porcine pancreatic alpha-amylase

Conversion of the substrate specificity of porcine pancreatic alpha-amylase (PPA) was studied using chemical modification of His residues. Diethyl pyrocarbonate modified His residues in PPA and the activity of the modified PPA for the hydrolysis of the alpha-D-(1,4)glucoside bond in starch or oligosaccharides decreased to less than 1% of that of the native enzyme. However, the activity for the hydrolysis of the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides was increased by chemical modification. When the modified PPA was incubated with a proteinaceous alpha-amylase inhibitor (Mr 60,000) purified from white kidney bean (Phaseolus vulgaris), it bound to the inhibitor. As a result, the remaining less than 1% hydrolytic activity of the modified PPA for starch disappeared completely but that for p-nitrophenyl oligosaccharides remained unaltered. The hydrolytic activity of the native PPA for the alpha-D-(1,4)glucoside bond in oligosaccharides was stronger than that between p-nitrophenyl and oligosaccharides in p-nitrophenyl oligosaccharides. Therefore, when p-nitrophenyl oligosaccharides (three to five glucose residues) were used as substrates for the native PPA, the alpha-D-(1,4)glucoside bonds in the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex hydrolyzed only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides. The above results reveal that, by chemical modification with diethyl pyrocarbonate and biochemical modification with an amylase inhibitor, amylase can be converted to a new exo-type enzyme which hydrolyzes only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides.     

无机离子和有机溶质对α-淀粉酶热稳定性的影响

长期以来,如何提高酶蛋白的热稳定性是分子生物学、生物工程学、化学工业等所关注的重要研究课题之一。分析了多种无机离子、糖和氨基酸对枯草杆菌液化型α-淀粉酶热稳定性的影响以及它们的共存效应,获取了一些对相关研究领域具有理论参考和实际应用价值的实验结果。在无机盐中,1mmol/L的钙离子或50mmol/L的钠离子能显著地提高该酶的热稳定性;酸性氨基酸和碱性氨基酸表现出相反的结果：酸性氨基酸具有明显的增强作用,碱性氨基酸却使之降低;随着糖浓度的增加（0～1000mmol/L）,该淀粉酶的热稳定性呈线性增高;当钠离子或钾离子与某些氨基酸或糖类共同存在时,对该淀粉酶的热稳定性表现出了明显的协同作用。试图通过检测酶蛋白分子荧光强度改变来反映该酶的热稳定性变化,其结果是：随着温度的改变,酶蛋白的荧光强度的衰减与残余酶活性之间显示了良好的相关性。从而说明热环境使酶蛋白分子的螺旋结构发生变化而失活,某些溶质的存在可能是通过作用于蛋白质分子的立体结构而影响该酶的热稳定性。     

1H NMR study of the base-pairing reactions of d(GGAATTCC): salt and polyamine effects on the imino proton exchange

Salts and polyamines have a variety of effects on the physical properties of DNA, including stabilization against thermal melting. We wished to gain greater insight into the mechanism of this stabilization by ascertaining its effect on the dynamics of base opening and closing reactions, as measured by NMR. Since the binding of spermidine(3+) is influenced by salt, and since spermidine may act as a base catalyst in proton exchange reactions, we have undertaken a study of salt and base catalyst effects on the imino proton exchange kinetics of a model oligomeric DNA. The selective longitudinal NMR relaxation rates of the hydrogen-bonded imino protons of the self-complementary octadeoxyribonucleotide d(GGAATTCC) monitor the rate of the base-catalyzed chemical exchange of these protons with solvent water. The exchange rates thus obtained provide a sensitive measure of the base-pair opening reactions of the DNA duplex. Under conditions of low pH and no added base catalyst, the NMR relaxation rates allow the determination of kd, the rate constant for the dissociation of the octameric duplex into single strands. Titration with the base catalyst tris(hydroxymethyl)aminomethane allows the determination of kop, the rate constant for the localized opening of individual base pairs, prior to dissociation. A significant Na+ concentration dependence is found for kd. From an analysis of this dependence, it is determined that 0.6 +/- 0.1 sodium ion is released during the dissociation event. The activation energy for helix dissociation (200 +/- 5 kJ/mol) is not dependent on the sodium ion concentration, indicating that the dissociation is entropically driven by the release of bound sodium ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural basis of alpha-amylase activation by chloride

To further investigate the mechanism and function of allosteric activation by chloride in some alpha-amylases, the structure of the bacterial alpha-amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to alpha-amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal alpha-amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.     

Pyrococcus furiosus alpha-amylase is stabilized by calcium and zinc

The hyperthermophilic archeon Pyrococcus furiosus produces an extracellular alpha-amylase that belongs to glycosyl hydrolases' family 13. This enzyme is more thermostable than its bacterial and archaeal homologues (e.g., Bacillus licheniformis TAKA-term and Pyrococcus kodakaraensis KOD1 alpha-amylases, respectively) even without adding Ca(2+) ions. Unlike the TAKA-therm amylase that contains no cysteine, the P. furiosus enzyme contains five cysteines (C152, C153, C165, C387, and C430), only four of which (C152, C153, C387, and C430) are conserved in the P. kodakaraensis alpha-amylase. To test the potential function of cysteines in P. furiosus alpha-amylase stability, these five residues were substituted with Ser or Ala-either one-by-one or in sequence-to produce eight mutant enzymes. Mutation C165S dramatically destabilized P. furiosus alpha-amylase. At the same time, the quadruple mutant enzyme C152S/C153S/C387S/C430A (mutant SSCSA) was as thermostable as the wild-type enzyme. Mutant SSCSA and wild-type alpha-amylases were strongly destabilized by dithiothreitol and ethylenediaminetetraacetic acid, suggesting that metal binding can be involved in this enzyme's thermostability. Inductively coupled plasma-atomic emission spectrometry showed the presence of Ca(2+) and Zn(2+) metal ions in P. furiosus alpha-amylase. Although Ca(2+) is known to contribute to alpha-amylase's stability, the absence of two out of the three conserved Ca(2+) ligands in the P. furiosus enzyme suggests that a different set of amino acids is involved in this enzyme's Ca(2+) binding. We also provide evidence suggesting that Cys165 is involved in Zn(2+) binding and that Cys165 is essential for the stability of P. furiosus alpha-amylase at very high temperatures.