Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown (original) (raw)
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Binding of glycogen, oligosaccharides, and glucose to glycogen debranching enzyme
Biochemistry, 1988
The binding of glucose and a series of oligosaccharides to glycogen debranching enzyme was determined by the ability of the saccharides to decrease the rate of reaction of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate) (DTNB). At pH 7.2, the strength of binding increases with chain length from glucose to maltotriose to maltopentaose but not to maltohexaose, and the free energies for binding of the oligosaccharides suggest subsites of equivalent affinities for the four glucose units following the initial reducing moiety. The rate of reaction of DTNB with enzyme saturated with saccharide is the same for all compounds, suggesting that all the saccharides, including gluose, induce the same conformational state. The site of binding may be that which binds the a-1,6-linked side chain of the natural limit dextrin substrate. At pH 8.0, this site exhibits similar characteristics, but an additional site, which may bind the four terminal glucose units of the main chain of the natural substrate, is manifested and exhibits different characteristics, including a very low affinity for glucose itself. The binding of glycogen to the debranching enzyme was monitored by centrifugal separation from the protein and exhibits a much lower dissociation constant than that for the oligomers, suggesting that branched polymers have more than one set of subsites. A m y lo-1,6-glucosidase/4-a-glucanotransferase (glycogen debranching enzyme) (EC 3.2.1.33 + EC 2.4.1.25) is a monomeric enzyme of about 165-kilodalton (kDa)' molecular mass that encompasses both glucosidase and transferase activities on a single polypeptide chain (Brown & Brown, 1966; Nelson et al., 1979). Following extensive studies with reversible inhibitors and a catalytic site directed irreversible inhibitor, Nelson and colleagues (Nelson et al., 1979; Gillard
Eur J Biochem, 2001
The hyperthermostable b-glycosidases from the Archaea Sulfolobus solfataricus (SsbGly) and Pyrococcus furiosus (CelB) hydrolyse b-glycosides of d-glucose or d-galactose with relaxed specificities pertaining to the nature of the leaving group and the glycosidic linkage. To determine how specificity is manifested under conditions of kinetically controlled transgalactosylation, the major transfer products formed during the hydrolysis of lactose by these enzymes have been identified, and their appearance and degradation have been determined in dependence of the degree of substrate conversion. CelB and SsbGly show a marked preference for making new b(133) and b(136) glycosidic bonds by intermolecular as well as intramolecular transfer reactions. The intramolecular galactosyl transfer of CelB, relative to glycosidic-bond cleavage and release of glucose, is about 2.2 times that of SsbGly and yields b-d-Galp-(136)-d-Glc and b-d-Galp-(133)-d-Glc in a molar ratio of < 1 : 2. The partitioning of galactosylated SsbGly between reaction with sugars [k Nu (m 21´s21 )] and reaction with water [k water (s 21 )] is about twice that of CelB. It gives a mixture of linear b-d-glycosides, chiefly trisaccharides at early reaction times, in which the prevailing new glycosidic bonds are b(136) and b(133) for the reactions catalysed by SsbGly and CelB, respectively. The accumulation of b-d-Galp-(136)-d-Glc at the end of lactose hydrolysis reflects a 3±10-fold specificity of both enzymes for the hydrolysis of b(133) over b(136) linked glucosides. Galactosyl transfer from SsbGly or CelB to d-glucose occurs with partitioning ratios, k Nu /k water , which are seven and . 170 times those for the reactions of the galactosylated enzymes with 1-propanol and 2-propanol, respectively. Therefore, the binding interactions with nucleophiles contribute chiefly to formation of new b-glycosides during lactose conversion. Likewise, noncovalent interactions with the glucose leaving group govern the catalytic efficiencies for the hydrolysis of lactose by both enzymes. They are almost fully expressed in the rate-limiting first-order rate constant for the galactosyl transfer from the substrate to the enzyme and lead to a positive deviation by < 2.5 log 10 units from structure±reactivity correlations based on the pK a of the leaving group. q FEBS 2000 Galactosyl transfer by thermostable b-glycosidases (Eur. J. Biochem. 267) 5057 q FEBS 2000 Galactosyl transfer by thermostable b-glycosidases (Eur. J. Biochem. 267) 5063 q FEBS 2000 Galactosyl transfer by thermostable b-glycosidases (Eur. J. Biochem. 267) 5065
Effects of oligosaccharide binding on glycogen debranching enzyme activity and conformation
Biochemistry, 1995
Glycogen debranching enzyme contains two catalytic activities (4-a-glucanotransferase and amylo-1,6-glucosidase) on its single polypeptide chain, and they are affected differently by the binding of oligosaccharides. Glucose, maltose, and maltotriose are competitive inhibitors of the amylo-1,6glucosidase activity measured by the hydrolysis of a-glucosyl fluoride, whereas saccharides with four or more glucose units are activators of the same activity, showing apparent "uncompetitive" kinetics. This suggests that they do not bind until the a-glucosyl fluoride is bound. In either case the potency of the effect increases with the length of the oligosaccharide chain. On the other hand, all oligosaccharides tested (maltose to maltohexaose, a-cyclodextrin, and P-cyclodextrin) are competitive inhibitors of the transferase activity and also cause a decrease in the intrinsic fluorescence, both functions again increased by chain length, thus indicating that these saccharides do bind to the free enzyme. These interesting results can be reconciled if the extended main chain resulting from the transferase reaction has to be reoriented into a different binding mode in order to position the a-l,6-linked side-chain glucose into the correct position for the glucosidase reaction. Therefore, activating oligosaccharides behave kinetically as if they had not been previously bound. It is concluded that the main chain of the natural limit dextrin substrate has a different mode of binding for the two catalytic reactions in order to position properly first the maltotetraosyl side chain in the transferase catalytic site and then the glucosyl side chain in the glucosidase catalytic site. All activating saccharides, including glycogen, elicit the same maximal glucosidase velocity, 6-fold the unactivated rate, suggesting that all generate the same enzyme conformation. Circular dichroic spectra yielded estimates of the secondary structure, but these were unaffected by any tested saccharide. Glycogen debranching enzyme (amylo-1,6-glucosidase/4a-glucanotransferase, EC 3.2.1.33 and EC 2.4.1.25) removes the 1,6-branch points in the limit dextrin resulting from the action of phosphorylase on glycogen. Its action consists of transferring a maltotriose unit to the "main chain" from the four glucose units attached to this main chain by a-1,6 linkages. This results in an elongated section of a-1,4-linked polymer, leaving a single glucose attached to it via an a-1,6 linkage. The enzyme then cleaves the a-1,6 linkage by which this glucose is attached. The transferase and glucosidase activities are both found on the single large polypeptide chain of this monomeric enzyme (Brown & Brown, 1966; Nelson et al., 1969; Bates et al., 1975), which for the rabbit enzyme has a molecular mass of 177 542 Da as estimated from its cDNA-derived protein sequence (Liu et al., 1993). Nelson and colleagues used reversible inhibitors and a catalytic site-directed irreversible inhibitor to analyze the two catalytic activities and concluded that the enzyme has
European Journal of Biochemistry, 2000
The hyperthermostable b-glycosidases from the Archaea Sulfolobus solfataricus (SsbGly) and Pyrococcus furiosus (CelB) hydrolyse b-glycosides of d-glucose or d-galactose with relaxed specificities pertaining to the nature of the leaving group and the glycosidic linkage. To determine how specificity is manifested under conditions of kinetically controlled transgalactosylation, the major transfer products formed during the hydrolysis of lactose by these enzymes have been identified, and their appearance and degradation have been determined in dependence of the degree of substrate conversion. CelB and SsbGly show a marked preference for making new b(133) and b(136) glycosidic bonds by intermolecular as well as intramolecular transfer reactions. The intramolecular galactosyl transfer of CelB, relative to glycosidic-bond cleavage and release of glucose, is about 2.2 times that of SsbGly and yields b-d-Galp-(136)-d-Glc and b-d-Galp-(133)-d-Glc in a molar ratio of < 1 : 2. The partitioning of galactosylated SsbGly between reaction with sugars [k Nu (m 21´s21 )] and reaction with water [k water (s 21 )] is about twice that of CelB. It gives a mixture of linear b-d-glycosides, chiefly trisaccharides at early reaction times, in which the prevailing new glycosidic bonds are b(136) and b(133) for the reactions catalysed by SsbGly and CelB, respectively. The accumulation of b-d-Galp-(136)-d-Glc at the end of lactose hydrolysis reflects a 3±10-fold specificity of both enzymes for the hydrolysis of b(133) over b(136) linked glucosides. Galactosyl transfer from SsbGly or CelB to d-glucose occurs with partitioning ratios, k Nu /k water , which are seven and . 170 times those for the reactions of the galactosylated enzymes with 1-propanol and 2-propanol, respectively. Therefore, the binding interactions with nucleophiles contribute chiefly to formation of new b-glycosides during lactose conversion. Likewise, noncovalent interactions with the glucose leaving group govern the catalytic efficiencies for the hydrolysis of lactose by both enzymes. They are almost fully expressed in the rate-limiting first-order rate constant for the galactosyl transfer from the substrate to the enzyme and lead to a positive deviation by < 2.5 log 10 units from structure±reactivity correlations based on the pK a of the leaving group. q FEBS 2000 Galactosyl transfer by thermostable b-glycosidases (Eur. J. Biochem. 267) 5057 q FEBS 2000 Galactosyl transfer by thermostable b-glycosidases (Eur. J. Biochem. 267) 5063 q FEBS 2000 Galactosyl transfer by thermostable b-glycosidases (Eur. J. Biochem. 267) 5065
Biochemistry (Moscow), 2013
Carbohydrate structures (glycostructures), such as glycoconjugates or oligo-and polysaccharide fragments, play numerous and often not fully understood roles in cellular processes. Glycostructures are involved in inflammation, intercellular interactions and signaling, immune response, viral and bacterial infections, energy accumulation, and in various matrix processes . Natural glycostructures are characterized by a great variety, but to study and understand their functions caused by the structural variability it is necessary to have significant amounts of the compound under study. Moreover, multiple biological activities of glycostructures determine the wide application of oligosaccharides with the desired composition in medicine and biotechnology . Traditional chemical approaches for synthesizing glycostructures consist of many time-consuming stages including procedures of blocking and deblocking of reactive chemical groups. Such syntheses, in addition to the desired product, give ecologically harmful waste. But for chemical synthesis of various carbohydrate-containing molecules there is an alternative, namely, enzymatic synthesis using two classes of enzymes, glycosyltransferases and glycoside hydrolases. However, the high price of nucleotide-activated sugars used as substrates for the directed synthesis of oligosaccharides with glycosyltransferases makes these enzymes less attractive. In turn, glycoside hydrolases capable of transglycosylating (transferring a carbohydrate substrate (donor) residue bound to the active site onto the hydroxyl of another sugar or alcohol (acceptor)) are promising tools for producing oligosaccharides . As a rule, these enzymes are stable, can be easily isolated, and their substrates are rather available. Glycoside hydrolases have already been used for synthesis of carbohydrate-containing structures for several dozens of years. Hundreds of papers concerning the synthesis of the glycosidic bond using glycoside hydrolases have been published (reviews ). Abstract-At high concentrations of p-nitrophenyl-α-D-galactopyranoside (pNPGal) as a substrate, its hydrolysis catalyzed by α-galactosidase from Thermotoga maritima (TmGalA) is accompanied by transglycosylation resulting in production of a mixture of (α1,2)-, (α1,3)-, and (α1,6)-p-nitrophenyl (pNP)-digalactosides. Molecular modeling of the reaction stage preceding the formation of the pNP-digalactosides within the active site of the enzyme revealed amino acid residues which modification was expected to increase the efficiency of transglycosylation. Upon the site-directed mutagenesis to the predicted substitutions of the amino acid residues, genes encoding the wild type TmGalA and its mutants were expressed in E. coli, and the corresponding enzymes were isolated and tested for the presence of the transglycosylating activity in synthesis of different pNP-digalactosides. Three mutants, F328A, P402D, and G385L, were shown to markedly increase the total transglycosylation as compared to the wild type enzyme. Moreover, the F328A mutant displayed an ability to produce a regio-isomer with the (α1,2)-bond at yield 16-times higher than the wild type TmGalA.
Journal of Microbiology and Biotechnology, 2019
We first confirmed the involvement of MalQ (4-α-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knockout mutant, ΔmalQ, showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-β-CD: G4-β-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.
Association of bi-functional activity in the N-terminal domain of glycogen debranching enzyme
Biochemical and Biophysical Research Communications, 2014
Glycogen debranching enzyme (GDE) in mammals and yeast exhibits a-1,4-transferase and a-1,6glucosidase activities within a single polypeptide chain and facilitates the breakdown of glycogen by a bi-functional mechanism. Each enzymatic activity of GDE is suggested to be associated with distinct domains; a-1,4-glycosyltransferase activity with the N-terminal domain and a-1,6-glucosidase activity with the C-terminal domain. Here, we present the biochemical features of the GDE from Saccharomyces cerevisiae using the substrate glucose(n)-b-cyclodextrin (Gn-b-CD). The bacterially expressed and purified GDE N-terminal domain (aa 1-644) showed a-1,4-transferase activity on maltotetraose (G4) and G4-b-CD, yielding various lengths of (G) n. Surprisingly, the N-terminal domain also exhibited a-1,6-glucosidase activity against G1-b-CD and G4-b-CD, producing G1 and b-CD. Mutational analysis showed that residues D535 and E564 in the N-terminal domain are essential for the transferase activity but not for the glucosidase activity. These results indicate that the N-terminal domain (1-644) alone has both a-1,4-transferase and the a-1,6-glucosidase activities and suggest that the bi-functional activity in the N-domain may occur via one active site, as observed in some archaeal debranching enzymes.
Biochemical and Biophysical Research Communications, 2008
Di-O-a-maltosyl-b-cyclodextrin ((G2) 2 -b-CD) was synthesized from 6-O-a-maltosyl-b-cyclodextrin (G2-b-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. TreX showed no activity toward glucosyl-b-CD, but a transfer product (1) was detected when the enzyme was incubated with maltosyl-b-CD, indicating specificity for a branched glucosyl chain bigger than DP2. Analysis of the structure of the transfer product (1) using MALDI-TOF/MS and isoamylase or glucoamylase treatment revealed it to be dimaltosyl-b-CD, suggesting that TreX transferred the maltosyl residue of a G2-b-CD to another molecule of G2-b-CD by forming an a-1,6-glucosidic linkage. When [ 14 C]-maltose and maltosyl-b-CD were reacted with the enzyme, the radiogram showed no labeled dimaltosyl-b-CD; no condensation product between the two substrates was detected, indicating that the synthesis of dimaltosyl-b-CD occurred exclusively via transglycosylation of an a-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (1) was identified to be isomers of 6 1 ,6 3 -and 6 1 ,6 4 -dimaltosyl-b-CD. Inhibition studies with b-CD on the transglycosylation activity revealed that b-CD was a mixed-type inhibitor, with a K i value of 55.6 lmol/mL. Thus, dimaltosyl-b-CD can be more efficiently synthesized by a transglycosylation reaction with TreX in the absence of b-CD. Our findings suggest that the high yield of (G2) 2 -b-CD from G2-b-CD was based on both the transglycosylation action mode and elimination of the inhibitory effect of b-CD.