Monte Carlo simulation of multiple attack mechanism of beta-amylase-catalyzed reaction

beta-Amylase (EC 3.2.1.2) produces maltose (dimer) from the nonreducing ends of alpha-1,4 glucosidic bonds of substrates like maltooligosaccharides, amylose, and amylopectin. The enzyme releases several maltose molecules from a single enzyme-substrate complex without dissociation by multiple or repetitive attack containing many branching reaction paths. The Monte Carlo method was applied to the simulation of the beta-amylase-catalyzed reaction including the multiple attack mechanism. The simulation starts from a single enzyme molecule and a finite number of substrate molecules. The selection of the substrate by the enzyme and degree of multiple attack proceeds by random numbers produced from a computer. The simulation was carried out until the whole substrate and the intermediate molecules were consumed. The simulated data were compared with experimental data of sweet potato beta-amylase using heptamer, octamer, nanomer, and 11-mer as substrates. The only adjustable parameter for odd-numbered substrates was the probability of multiple attack, while an additional adjustable parameter (a correction factor due to low reactivity of tetramer) was needed for even-numbered substrates.     

Hydrolysis of amylopectin by amylolytic enzymes: structural analysis of the residual amylopectin population

Amylopectin fine structures were studied following limited hydrolysis of gelatinised waxy maize starch by amylases with a different level of inner chain attack (LICA). This was done by size exclusion chromatography as well as by debranching the (partially hydrolysed) amylopectin samples and studying the size distributions of the released chains. α-Amylases from Bacillus amyloliquefaciens and Aspergillus oryzae, with a relatively high LICA, drastically altered amylopectin chain length distribution and reduced the amylopectin molecular size (MS) significantly even at a low to moderate degree of hydrolysis (DH). Porcine pancreatic α-amylase (PPA), with a rather low LICA but a high multiple attack action on amylose, reduced the amylopectin MS much slower. Following hydrolysis by PPA to a DH of 10% and enzymic debranching of the amylopectin residue, several subpopulations of chains consisting of 2-12 glucose units were detected, indicating a multiple attack action on the amylopectin side chains. During the early stages of hydrolysis, the maltogenic Bacillus stearothermophilus α-amylase (BStA) preferentially hydrolysed the exterior chains of amylopectin. However, during the later phases, BStA also hydrolysed inner chains, presumably with a high multiple attack action. The present results clearly show that different enzymes can be used for (limited) conversion of amylopectin into structures differing in molecular weight and chain length distributions.     

Multiple attack in porcine pancreatic alpha-amylase-catalyzed hydrolysis of amylose studied with a fluorescence probe.

1. A large fluorescence enhancement of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) observed in the presence of amylose was utilized to monitor quantitatively the time course of porcine pancreatic alpha-amylase [EC 3.2.1.1] (PPA)-catalyzed hydrolysis of amylose with a number-average degree of polymerization of 16.8. 2. The slope of the plot of decrease in the relative fluorescence intensity of the TNS-amylose system (termed as the fluorescence value) versus the number of linkages hydrolyzed (reducing value) (Kondo, H. et al. (1977) Agric. Biol. Chem. 41, 631-634) in the course of PPA-catalyzed hydrolysis was shown to be useful to describe the degree of "multiple attack," which is defined by the number of reattacks on a long chain substrate molecular per one encounter of the enzyme and the substrate. A parameter gamma was defined as the ratio of the reciprocal of the slopes obtained at each pH to that at pH 10.5, where the multiple attack is not operating. 3. The gamma versus pH profile gave an apparent pK value of about 9, indicating that some ionizable groups participate in the multiple attack mechanism. 4. Based on a reaction scheme involving a "sliding" of the substrate molecule on the enzyme, which may contribute to the multiple attack mechanism, besides binding, dissociation, and cleavage steps of the substrate, and on the assumption of the steady state for the enzyme-substrate complex, rate equations were obtained to describe the time course of hydrolysis of a linear substrate. The product distribution with the progress of the reaction can be calculated theoretically, and is dependent on the number of multiple attack and the mode of sliding. The number of multiple attack can be estimated from this distribution, and the fluorescence value can be calculated theoretically by combining the product distribution with the relative efficiency of fluorescence intensity of each maltooligosaccharide (Nakatani, H. et. al. (1977) Biopolymers 16, 2363-2370). By comparing the experimental data with the theoretical ones, it was suggested that the multiple attack occurs through the sliding by maltose unit of the retained fragment on the enzyme, which is one of the fragments produced by the initial cleavage of the substrate molecule. 5. It was found that anions (chloride, bromide, and nitrate ions) which critically affect the enzyme activity have no effect on the degree of multiple attack.     

Purification and properties of potato 1,4-alpha-D-glucan:1,4-alpha-D-glucan 6-alpha-(1,4-alpha-glucano)-transferase. Evidence against a dual catalytic function in amylose-branching enzyme.

Q-Enzyme, the enzyme that synthesizes the 1,6-alpha-glucosidic branch linkages of amylopectin, has been purified from potato to near homogeneity. The molecular weight of the enzyme is 85000. The active enzyme is a monomer, with a molar activity at pH 7.0 and 24 degrees C of 15. The energy of activation is 25 kJ/mol below 15 degrees C, changing sharply to 63 kJ/mol above that temperature. Enzyme activity is not affected by Mg2+ or ATP. There are about 11 readily titratable sulfhydryl groups per molecule. The evidence that the enzyme is a single protein entity, without hydrolytic activity towards amylose, contrasts with an earlier report that Q-enzyme consists of two components, a hydrolase with molecular weight 70000, and a transferase with molecular weight 20000. Q-enzyme acts on native and synthetic amyloses to give products resembling amylopectin in terms of average unit chain length, degress of beta-amylolysis and iodine stain. The profiles of the unit chains of these synthetic products are, however, different from that of native amylopectin. Additional branch linkages are introduced by Q-enzyme into potato amylopectin, but the product bears no resemblance to phytoglycogen.     

Genotype-specific spatial distribution of starch molecules in the starch granule: a combined CLSM and SEM approach

Starch granule types from a variety of botanical sources were selected to represent differences in crystalline polymorph, amylose and phosphate content, and amylopectin chain length distribution. Equimolar labeling of starch molecules with the fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) was used to construct a detailed map of the distribution of amylose and amylopectin within the granule by confocal laser scanning microscopy (CLSM) analysis. Medium- and high-resolution scanning electron microscopy (SEM) were used to provide detailed images of granule surface structures. By using a combined surface and internal imaging approach, interpretations of a number of previous structural observations is presented. In particular, internal images of high amylose maize and potato suggest that multiple initiations of new granules are responsible for the compound or elongated structures observed in these starches. CLSM optical sections of rice granules revealed an apparent altered distribution of amylose in relation to the proposed growth ring structure, hinting at a novel mechanism of starch molecule deposition. Well-described granule features, such as equatorial grooves, channels, cracks, and growth rings were documented and related to both the internal and external observations. A new method for probing the phosphate distribution in native granules was developed using a phosphate-binding fluorescent dye and CLSM.     

Physico-chemical and degradative properties of in-planta re-structured potato starch

Starch re-structured directly in potato tubers by antisense suppression of starch branching enzyme (SBE), granule bound starch synthase (GBSS) or glucan water dikinase (GWD) genes was studied with the aim at disclosing the effects on resulting physico-chemical and enzyme degradative properties. The starches were selected to provide a combined system with specific and extensive alterations in amylose and covalently esterified glucose-6-phosphate (G6P) contents. As an effect of the altered chemical composition of the starches their hydrothermal characteristics varied significantly. Despite of the extreme alterations in phosphate content, the amylose content had a major affect on swelling power, enthalpy for starch gelatinization and pasting parameters as assessed by Rapid Visco Analysis (RVA). However, a combined influence of the starch phosphate and long glucan chains as represented by high amylose or long amylopectin chain length was indicated by their positive correlation to the final viscosity and set back (RVA) demonstrating the formation of a highly hydrated and gel-forming system during re-structuring of the starch pastes. Clear inverse correlations between glucoamylase-catalyzed digestibility and amylopectin chain length and starch phosphate and lack of such correlation with amylose content indicates a combined structuring role of the phosphate groups and amylopectin chains on the starch glucan matrix.     

Conformational Dynamics of Human AP Endonuclease in Base Excision and Nucleotide Incision Repair Pathways

Abstract

APE1 is a multifunctional enzyme that plays a central role in base excision repair (BER) of DNA. APE1 is also involved in the alternative nucleotide incision repair (NIR) pathway. We present an analysis of conformational dynamics and kinetic mechanisms of the full-length APE1 and truncated NΔA61-APE1 lacking the N-terminal 61 amino acids (REF1 domain) in BER and NIR pathways. The action of both enzyme forms were described by identical kinetic schemes, containing four stages corresponding to formation of the initial enzyme-substrate complex and isomerization of this complex; when a damaged substrate was present, these stages were followed by an irreversible catalytic stage resulting in the formation of the enzyme-product complex and the equilibrium stage of product release. For the first time we showed, that upon binding AP-containing DNA, the APE1 structure underwent conformational changes before the chemical cleavage step. Under BER conditions, the REF1 domain of APE1 influenced the stability of both the enzyme-substrate and enzyme-product complexes, as well as the isomerization rate, but did not affect the rates of initial complex formation or catalysis. Under NIR conditions, the REF1 domain affected both the rate of formation and the stability of the initial complex. In comparison with the full-length protein, NΔA61-APE1 did not display a decrease in NIR activity with a dihydrouracil-containing substrate. BER conditions decrease the rate of catalysis and strongly inhibit the rate of isomerization step for the NIR substrates. Under NIR conditions AP-endonuclease activity is still very efficient.     

Inhibition Pattern of Beet-Saponin on Amylose Formation by Potato Phosphorylase

By using the multiple ascent technique, the authors have resolved the first several oligosaccharides of the product of initial stage of potato phosphorylase action in both the absence and the presence of beet-saponin with maltotriose as primer. The resolved chromatogram was sprayed with a mixture of G-1-P and phosphorylase, followed by spraying with iodine solution to locate the spots in which starch synthesis occurred.

Multi-chain mechanism of amylose formation from maltotriose and the suppression of lengthening of amylose chain by beet-saponin in the lag stage of enzyme action could be shown on paper chromatogram. No saccharides other than amylose series were recognized in the case of phosphorylase inhibition by beet-saponin.     

Temperature impacts the multiple attack action of amylases

The action pattern of several amylases was studied at 35, 50, and 70 degrees C using potato amylose, a soluble (Red Starch) and insoluble (cross-linked amylose) chromophoric substrate. With potato amylose as substrate, Bacillus stearothermophilus alpha-amylase (BStA) and porcine pancreatic alpha-amylase displayed a high degree of multiple attack (DMA, i.e., the number of bonds broken during the lifetime of an enzyme-substrate complex minus one), the fungal alpha-amylase from Aspergillus oryzae a low DMA, and the alpha-amylases from B. licheniformis, Thermoactinomyces vulgaris, B. amyloliquifaciens, and B. subtilis an intermediate DMA. These data are discussed in relation to structural properties of the enzymes. The level of multiple attack (LMA), based on the relation between the drop in iodine binding of amylose and the increase in total reducing value, proved to be a good alternative for DMA measurements. The LMA of the endo-amylases increased with temperature to a degree depending on the amylase. In contrast, BStA showed a decreased LMA when temperature was raised. Furthermore, different enzymes had different activities on Red Starch and cross-linked amylose. Hence, next to the temperature, the action pattern of alpha-amylases is influenced by structural parameters of the starch substrate.     

Mixture design of starchy substrates hydrolysis by an immobilized glucoamylase from Aspergillus brasiliensis

Starch has great importance in human diet, since it is a heteropolymer of plants, mainly found in roots, as potato, cassava and arrowroots. This carbohydrate is composed by a highly-branched chain: amylopectin; and a linear chain: amylose. The proportion between the chains varies according to the botanical source. Starch hydrolysis is catalyzed by enzymes of the amilolytic system, named amylases. Among the various enzymes of this system, the glucoamylases (EC 3.2.1.3 glucan 1,4-alpha-glucosidases) are the majority because they hydrolyze the glycosidic linkages at the end of starch chains releasing glucose monomers. In this work, a glucoamylase secreted in the culture medium, by the ascomycete Aspergillus brasiliensis, was immobilized in Dietilaminoetil Sepharose-Polyethylene Glycol (DEAE-PEG), since immobilized biocatalysts are more stable in long periods of hydrolysis, and can be recovered from the final product and reused for several cycles. Glucoamylase immobilization has shown great thermal stability improvement over the soluble enzyme, reaching 66% more activity after 6?h at 60?°C, and 68% of the activity after 10 hydrolysis cycles. A simplex centroid experimental mixture design was applied as a tool to characterize the affinity of the immobilized enzyme for different starchy substrates. In assays containing several proportions of amylose, amylopectin and starch, the glucoamylase from A. brasiliensis mainly hydrolyzed the amylopectin chains, showing to have preference by branched substrates.     

Analysis of ping-pong reaction mechanisms by positional isotope exchange. Application to galactose-1-phosphate uridyltransferase

A new positional isotope exchange method has been developed that can be used for the analysis of enzyme-catalyzed reactions which have ping-pong kinetic mechanisms. The technique can be used to measure the relative rates of ligand dissociation from enzyme-product complexes. Enzyme is incubated with the labeled substrate and an excess of the corresponding unlabeled product. The partitioning of the enzyme-product complex back toward free enzyme is determined from the rate of positional isotope exchange within the original labeled substrate. The partitioning of the enzyme-product complex forward toward free enzyme is determined from the rate of formation of totally unlabeled substrate. It has been shown that the ratio of the two rates provides a lower limit for the release of product from the enzyme-product complex. The technique has been applied to the reaction catalyzed by galactose-1-phosphate uridyltransferase. The lower limit for the release of glucose 1-phosphate from the uridyl-enzyme relative to the maximal velocity of the reverse reaction was determined to be 3.4 +/- 0.5.     

Production of tailor made short chain amylose–lipid complexes using varying reaction conditions

When amylose was synthesized using potato phosphorylase in the presence of amylose complexing lipids, monodisperse populations of amylose–lipid complexes were formed. Enzyme dosage and glucose-1-phosphate (glc-1-P)/primer ratio influenced the reaction rate of the enzymic synthesis, presumably by changing the balance between amylose synthesis and amylose–lipid complexation and precipitation, and impacted the molecular weight of the complexes. Lipid characteristics affected the dissociation properties and amylose chain lengths of the amylose–lipid complexes presumably by determining the minimal amylose chain length necessary for complexation and precipitation. Tailor made short chain amylose–lipid complexes can hence be produced by choosing the appropriate reaction conditions. We propose a synthesis mechanism in which the primer is elongated until an amylose chain is obtained which is of sufficient length to complex a first lipid. Further chain extension then occurs, together with subsequent complexation until the complex becomes insoluble and precipitates.     

Importance of isoforms of starch-branching enzyme in determining the structure of starch in pea leaves

The impact of a mutation at the r locus of peas ( Pisum sativum L.) on the structure of starch in the leaf has been studied. The mutation specifically eliminates the A class of isoform of starch-branching enzyme (SBE A) from the leaf, causing a 10-fold reduction in the total activity of the enzyme. Gel-permeation chromatography and thymol precipitation show that wild-type leaf starch consists of polymers with the general characteristics of amylose and amylopectin, although amylose is only a very minor component of the starch. High-performance anion exchange chromatography (HPAEC) of debranched amylopectin reveals that the distribution profile of branch lengths is strongly polymodal, and distinctly different from that of the amylopectin of storage starches. The mutation at the r locus results in the appearance of an amylopectin-like glucan of low molecular weight in the starch. The absorbance of the iodine complex of the amylopectin and analysis by HPAEC both indicate that the mutation causes an increase in the average branch length of the amylopectin but does not affect the polymodal nature of the distribution of branch lengths. The extent to which these effects of the mutation are specifically due to the loss of SBE A is discussed. It is suggested that differences in properties between isoforms of SBE are not the main factors that determine the polymodal distribution of branch lengths in amylopectin.     

Monte Carlo simulation of 4‐α‐glucanotransferase reaction

4‐α‐Glucanotransferase (GTase, D ‐enzyme) catalyzes disproportionation between two short polymers of maltooligosaccharides linked by α‐1,4‐glucoside bonds. Using action modes of the potato GTase for the donor and acceptor substrates, the Monte Carlo method was applied to simulate the GTase reaction. The simulation starts from a single enzyme molecule and a finite number (10⁵) of substrate molecules. All selection processes were performed using random numbers produced by computer. The initial substrates were from trimer to 10‐mer. In every case, the final stage was the steady‐state distribution of polymers. The steady‐state distribution by the potato GTase reaction was different from those by the hypothetical random disproportionation reaction. The simulated data from the reaction of potato GTase and trimer almost quantitatively agreed with experimental data. The mechanism of the GTase reaction was accumulation of probabilistic processes and was well simulated by the Monte Carlo method. GTase randomizes the overall distribution of chain length of the substrate. Therefore the GTase reaction is an entropy‐driven process. © 1999 John Wiley & Sons, Inc. Biopoly 50: 145–151, 1999     

Pathway of cytosolic starch synthesis in the model glaucophyte Cyanophora paradoxa

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model glaucophyte Cyanophora paradoxa. The storage polysaccharide granules are shown to be composed of both amylose and amylopectin fractions, with a chain length distribution and crystalline organization similar to those of green algae and land plant starch. A preliminary characterization of the starch pathway demonstrates that Cyanophora paradoxa contains several UDP-glucose-utilizing soluble starch synthase activities related to those of the Rhodophyceae. In addition, Cyanophora paradoxa synthesizes amylose with a granule-bound starch synthase displaying a preference for UDP-glucose. A debranching enzyme of isoamylase specificity and multiple starch phosphorylases also are evidenced in the model glaucophyte. The picture emerging from our biochemical and molecular characterizations consists of the presence of a UDP-glucose-based pathway similar to that recently proposed for the red algae, the cryptophytes, and the alveolates. The correlative presence of isoamylase and starch among photosynthetic eukaryotes is discussed.     

On the mechanism of amylose branching by potato Q-enzyme.

1. When potato Q-enzyme converts amylose into an amylopectin-like molecule, the action is by a random, endo-type transglycosylation of the substrate chains. 2. Inter-chain transfer takes place during the formation of the amylopectin branch linkage. This is seen in experiments in which radioactive label was transferred between substrates of disparate molecular weight. Intra-chain transfer, leading to the formation of a branch linkage, is not excluded by these experiments. 3. The minimum length of amylose chain that can act as an acceptor in the transglycosylation reaction, under the experimental conditions described, is greater than 40 glucose units. 4. The requirement of Q-enzyme for substrate chains at least 40 glucose units in length is interpreted as meaning that a stabilized secondary and tertiary structure must be established in the substrate before it can be utilized by Q-enzyme, and that the forces that provide such conformation are sufficiently strong only when the chains are longer than the minimum. Inter-chain transfer is seen as taking place by one of two mechanisms. The first involved the reaction of the enzyme with a chain that has a stabilized (helical?) conformation. An enzyme-donor chain intermediate is formed, that then reacts with an acceptor chain to complete the transglycosylation. The second mechanism envisages the substrate for the enzyme as being a complex formed between two chains (a double helix?). The enzyme encounters the complex and carries out an inter-chain transglycosylation reactions.     

Size Distribution of Dispersing Units in Corn Amylose

As the first step to reveal the size distribution of dispersing units in amylose, an amylose sample from corn starch was subjected to gel-filtration chromatography on columns of Sephadex G 200 and Sepharoses 6 B, 4 B and 2 B under protection of the sample from retro-gradation by the use of thiocyanate in the solvent system in the chromatography,

A considerable amount of dispersing units with unexpectedly large size was detected in the amylose sample as a. fraction which was excluded from gels of Sepharoses, though the size distribution was considered to be rather continuous covering the units with smaller size of the regular amylose in the sample. The excluded fraction involved an appreciable amount of aggregates of amylose but no usual amylopectin. Nevertheless, the fraction involved some α-1,6 glucosidic linkages susceptible to isoamylase. The digest of the fraction by the enzyme gave a product which showed size distribution similar to that of the regular size of amylose in the original sample. Both the digested product and the regular amylose had much larger size in molecular weight than the digest of amylopectin by the same enzyme.

The results indicate that the corn amylose sample is a mixture of the units with regular size amylose and those with very large one, the latter having various numbers of branches with the size of regular amylose. Thus, it is considered that the size of dispersing units in the corn amylose sample is characterized as a pattern of such continuous distribution ranging from few hundred thousand up to more than several million in terms of molecular weight.     

Stopped-flow studies on the mechanism of oxidation of N-methyl-4-phenyltetrahydropyridine by bovine liver monoamine oxidase B

The kinetic mechanism of monoamine oxidase B involves either a binary or a ternary complex, depending on the substrate. In this study, stopped-flow kinetic data provide direct evidence for ternary complexes not only of reduced enzyme, oxygen, and product but also of reduced enzyme, oxygen, and substrate, both for benzylamine and for the tertiary amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the mechanism for a given substrate is not exclusive but, rather, is determined by competition between the alternate pathways as a result of different rate constants for the oxidation of the reduced enzyme, the reduced enzyme-product complex, and the reduced enzyme-substrate complex, as well as the different dissociation constants for the complexes. Comparison of the rate constants obtained from the stopped-flow studies with steady-state data indicates that the overall rate of reaction for the oxidation of MPTP by monoamine oxidase is dominated by the reductive step, but for benzylamine the steady-state rate is determined by a complex function of the rates of both the reductive and oxidative half-reactions.     

Characterization of barley starches of waxy, normal, and high amylose varieties

Four varieties of barley starches, W.B. Merlin, glacier, high amylose glacier, and high amylose hull-less glacier, were isolated from barley seeds. Apparent and absolute amylose contents, molecular size distributions of amylose and amylopectin, amylopectin branch-chain-length distributions, and Naegeli dextrin structures of the starches were analyzed. W.B. Merlin amylopectin had the longest detectable chain length of DP 67, whereas glacier, high amylose glacier and high amylose hull-less glacier amylopectins had the longest detectable chain length of DP 82, 79, and 78, respectively. All the four starches displayed a substantially reduced proportion of chains at DP 18–21. Amylopectins of high amylose varieties did not show significantly larger proportions of long chains than that of normal and waxy barley starch. Onset gelatinization temperatures of all four barley starches ranged from 55.0 to 56.5°C. Absolute amylose contents of W.B. Merlin, glacier, high amylose glacier, and high amylose hull-less glacier were 9.1, 29.5, 44.7, and 43.4%, respectively; phospholipid contents were 0.36, 0.78, 0.79, and 0.97%, respectively.     

Gel formation in mixtures of high amylopectin potato starch and potato starch

The effects of starch granules on the rheological behaviour of gels of native potato and high amylopectin potato (HAPP) starches have been studied with small deformation oscillatory rheometry. The influence of granule remnants on the rheological properties of samples treated at 90 °C was evident when compared with samples treated at 140 °C, where no granule remnants were found. The presence of amylose in native potato starch gave to stronger network formation since potato starch gave higher moduli values than HAPP, after both 90 and 140 °C treatments. In addition, amylose may have strengthened the network of HAPP because higher moduli values were obtained when native potato starch was added to the system. The moduli values of the mixtures also increased with increasing polysaccharide concentration in the system, which is due to an increment in the polysaccharide chain contacts and entanglements. Finally, it was found that a mixture of commercial amylose from potato starch and HAPP resulted in lower values of G′ compared to native potato starch. This indicates that the source of amylose is important for the properties in a blend with native amylopectin.