Kinetics of formation of maltose and isomaltose through condensation of glucose by glucoamylase

A kinetic model was devised for the hydrolysis and synthesis of maltose and isomaltose by two glucoamylases from Rhizopus niveus and Aspergillus niger, and the validity of the model was verified experimentally at 313 K and pH 5.0. For both enzymes, the formations of maltose and isomaltose from glucose were parallel reversible reactions, and glucosyl transfer between maltose and isomaltose was not observed. The enzymes catalyzed rapid hydrolysis and synthesis of maltose. Isomaltose was hydrolyzed and synthesized more slowly, but the level produced from glucose was much higher than that of maltose. These hydrolysis and condensation reactions were expressed well by the model.     

Inhibition by maltose, isomaltose, and nigerose of the synthesis of high-molecular-weight D-glucans by the D-glucosyltransferases of Streptococcus sobrinus

Two D-glucosyltransferases are produced by Streptococcus sobrinus C211. One (GTF-S) catalyzes the conversion of sucrose into soluble alpha-(1----6)-linked alpha-(1----3)-branched D-glucans, and the other (GTF-I), of sucrose into alpha-(1----3)-linked alpha-(1----6)-branched D-glucans. These enzymes were studied by using maltose, isomaltose, and nigerose as inhibitors. Maltose and isomaltose were found to be competitive inhibitors of GTF-S, whereas nigerose has no effect on GTF-S activity. The Ki values for maltose and isomaltose were determined to be 11 and 15mM, respectively. Maltose, isomaltose, and nigerose competitively inhibit GTF-I. The Ki values for these inhibitors were found to be approximately 0.8, 2.5, and 15mM, respectively. The inhibitory properties of each disaccharide are interpreted in terms of conformational comparisons with sucrose.     

A kinetic model for the hydrolysis and synthesis of maltose, isomaltose, and maltotriose by glucoamylase

Kinetic results on the glucomylase-catalysed hydrolysis of maltose and maltotriose, and glucose polymerization into maltose and isomaltose up to 450 g/L total sugar concentration are presented. Whereas the enzyme has a faster hydrolytic and synthetic activity on alpha-(1-->4) than on alpha-(1-->6) linkages, at equilibrium, on the contrary, the isomaltose level which represents 15% (w/w) of the total sugar concentration at the highest investigated concentrations is much higher than the corresponding maltose level. Under a wide range of initial conditions, experimental results are adequately described by a new kinetic model with simple first- and second-order, or Michaelian-type, rate expressions for the reversible hydrolysis of maltotriose, maltose, and isomaltose. The model also accounts for the inhibition of hydrolysis by glucose, but does not consider the concentration of water which, under the present conditions, was not found kinetically limiting.     

Molecular and physiological role of the trehalose-hydrolyzing alpha-glucosidase from Thermus thermophilus HB27

Trehalose supports the growth of Thermus thermophilus strain HB27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. We expressed a putative alpha-glucosidase gene (TTC0107), characterized the recombinant enzyme, and found that the preferred substrate was alpha,alpha-1,1-trehalose, a new feature among alpha-glucosidases. The enzyme could also hydrolyze the disaccharides kojibiose and sucrose (alpha-1,2 linkage), nigerose and turanose (alpha-1,3), leucrose (alpha-1,5), isomaltose and palatinose (alpha-1,6), and maltose (alpha-1,4) to a lesser extent. Trehalose was not, however, a substrate for the highly homologous alpha-glucosidase from T. thermophilus strain GK24. The reciprocal replacement of a peptide containing eight amino acids in the alpha-glucosidases from strains HB27 (LGEHNLPP) and GK24 (EPTAYHTL) reduced the ability of the former to hydrolyze trehalose and provided trehalose-hydrolytic activity to the latter, showing that LGEHNLPP is necessary for trehalose recognition. Furthermore, disruption of the alpha-glucosidase gene significantly affected the growth of T. thermophilus HB27 in minimal medium supplemented with trehalose, isomaltose, sucrose, or palatinose, to a lesser extent with maltose, but not with cellobiose (not a substrate for the alpha-glucosidase), indicating that the alpha-glucosidase is important for the assimilation of those four disaccharides but that it is also implicated in maltose catabolism.     

Purification and characterization of two phosphoglucomutases from Lactococcus lactis subsp. lactis and their regulation in maltose- and glucose-utilizing cells.

Two distinct forms of phosphoglucomutase were found in Lactococcus lactis subsp. lactis, strains 19435 and 65.1, growing on maltose: beta-phosphoglucomutase (beta-PGM), which catalyzes the reversible conversion of beta-glucose 1-phosphate to glucose 6-phosphate in the maltose catabolism, and alpha-phosphoglucomutase (alpha-PGM). beta-PGM was purified to more than 90% homogeneity in crude cell extract from maltose-grown lactococci, and polyclonal antisera to the enzyme were prepared. The molecular mass of beta-PGM was estimated by gel filtration to be 28 kDa; its isoelectric point was 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The expression of both PGM enzymes was investigated under different growth conditions. The specific activity and amount of beta-PGM per milliliter of cell extract increased with time in lactococci grown on maltose, but the enzyme was absent in lactococci grown on glucose, indicating enzyme synthesis to be induced by maltose in the growth medium. When glucose was added to maltose-grown lactococci, both the specific activity and amount of beta-PGM per milliliter of cell extract decreased rapidly. This suggests that synthesis of beta-PGM is repressed by glucose in the medium. Although the specific activity of alpha-PGM did not change during growth on maltose or glucose, lactococcal strain 19435 showed a much higher specific activity of both alpha- and beta-PGM than strain 65.1 when grown on maltose.     

Purification and characterization of cyclic maltosyl-(1-->6)-maltose hydrolase and alpha-glucosidase from an Arthrobacter globiformis strain

Cyclic maltosyl-maltose [CMM, cyclo-[-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate alpha-1,4 and alpha-1,6 linkages. CMM is synthesized from starch by the action of 6-alpha-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and alpha-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 degrees C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 degrees C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the alpha-1,6-linkage of CMM. The molecular mass of alpha-glucosidase was determined to be 60.1 kDa by SDS-PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for alpha-glucosidase activity were 7.0 and 35 degrees C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 degrees C. alpha-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and alpha-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and alpha-glucosidase.     

Differential inhibition of branching enzyme in a morphological mutant and in wild type Neurospora. Influence of carbon source in the growth medium.

1. A morphological mutant of Neurospora crassa, smco 9, (R2508) that exhibits colonial morphology when grown on sucrose or on maltose, showed a partial reversal of this morphology toward that of the wild type when it was grown on potato starch or on isomaltose. 2. A common feature of both potato starch and isomaltose is the presence of alpha-1, 6 glucosidic linkages. This suggested that these morphological effects might be due to differences in alpha-1,4 glucan: alpha-1,4 glucan 6 glycosyltransferase, (EC 2.4.1.18) commonly known as "the branching enzyme". 3. The branching enzyme was purified from wild type, Neurospora crassa, and from the semicolonial mutant, R2508, both grown on sucrose or on potato starch. It has a molecular weight of 140,000 as estimated by gel filtration on a Bio Gel A 1.5 m column. This enzyme plus phosphorylase a in an unprimed reaction catalyzes the synthesis of a branched polysaccharide in vitro. 4. No branching enzyme activity was apparent in extracts of the mutant R2508, grown on potato starch until a thermolabile inhibitor was removed by fractionation on a DEAE column. 5. This inhibitor has a molecular weight greater than 100,000 as estimated on a P-100 polyacrylamide gel column. The specificity of the inhibitor is not absolute in that it inhibits glycogen synthetase in addition to the branching enzyme in Neurospora.     

Effects of alpha-D-glucosylglycerol on the in vitro digestion of disaccharides by rat intestinal enzymes

Alpha-D-glucosylglycerol (GG) is a mixture of 2-O-alpha-D-glucosylglycerol (GG-II), (2R)-1-O-alpha-D-glucosylglycerol (R-GG-I) and (2S)-1-O-alpha-D-glucosylglycerol (S-GG-I). GG has been found to be slightly hydrolyzed in vitro only by rat intestinal enzymes, but hardly at all by other digestive juices. GG suppressed the hydrolysis of maltose, sucrose and isomaltose by rat intestinal enzymes because the amount of glucose in the digestion of a mixture of GG and disaccharide was less than the sum of that in each individual digestion. The consumption of GG was suppressed by isomaltose, but promoted by maltose, with the hydrolysis of GG being suppressed. Sucrose appeared to suppress only the consumption of S-GG-I, suggesting that S-GG-I was hydrolyzed by the active site of sucrase in a sucrase-isomaltase complex. Transglucosylation seems to have occurred more frequently in the individual digestion of maltose and isomaltose than in that of GG and sucrose. GG seemed to promote transglucosylation in the presence of maltose, to suppress it with sucrose, and to delay it with isomaltose.     

A novel alpha-glucosidase from the moss Scopelophila cataractae

Scopelophila cataractae is a rare moss that grows on copper-containing soils. S. cataractae protonema was grown on basal MS medium containing copper. A starch-degrading activity was detected in homogenates of the protonema, after successive extraction with phosphate buffer and buffer containing 3 M LiCl. Buffer-soluble extract (BS) and LiCl-soluble extract (LS) readily hydrolyzed amylopectin to liberate only glucose, which shows that alpha-glucosidase (EC 3.2.1.20) in BS and LS hydrolyzed amylopectin. The K(m) value of BS for maltose was 0.427. The K(m) value of BS for malto-oligosaccharide decreased with an increase in the molecular mass of the substrate. The value for maltohexaose was 0.106, which is about four-fold lower than that for maltose. BS was divided into two fractions of alpha-glucosidase (BS-1 and BS-2) by isoelectric focusing. The isoelectric points of these two enzymes were determined to be 4.36 (BS-1) and 5.25 (BS-2) by analytical gel electrofocusing. The two enzymes readily hydrolyzed malto-oligosaccharides. The two enzymes also hydrolyzed amylose, amylopectin and soluble starch at a rate similar to that with maltose. The two enzymes readily hydrolyzed panose to liberate glucose and maltose (1 : 1), and the K(m) value of BS for panose was similar to that for maltotriose, whereas the enzymes hydrolyzed isomaltose only weakly. With regard to substrate specificity, the two enzymes in BS are novel alpha-glucosidases. The two enzymes also hydrolyzed beta-limit dextrin, which has many alpha-1,6-glucosidic linkages near the non-reducing ends, more strongly than maltose, which shows that they do not need a debranching enzyme for starch digestion. The starch-degrading activity of BS was not inhibited by p-chloromercuribenzoic acid or alpha-amylase inhibitor. When amylopectin was treated with BS and LS in phosphate buffer, pH 6.0, glucose, but not glucose-1-phosphate, was detected, showing that the extracts did not contain phosphorylase but did contain an alpha-glucosidase. These results show that alpha-glucosidases should be capable of complete starch digestion by themselves in cells of S. cataractae.     

Kinetics of condensation of glucose into maltose and isomaltose in hydrolysis of starch by glucoamylase

Kinetics of the condensation of glucose into maltose and isomaltose in the hydrolysis of starch by two types of glucoamylase (from Aspergillus niger and Rhizopus niveus) was studied both experimentally and theoretically. A kinetic model for the hydrolysis of starch by glucoamylase from A. niger was proposed. In this model the reversible hydrolysis of maltose and isomaltose and the kinetic parameters change were taken into consideration. Calculated values agreed approximately with the experimental results, and this simple kinetic model was found to have practical use. The rate of condensation of glucose into isomaltose by enzyme from A. niger was about three times larger than that by enzyme from R. niveus. At a higher initial concentration of starch a large amount of isomaltose was reversed, and the glucose yield was reduced significantly after very long reaction times.     

Resistance to quaternary ammonium compounds in Staphylococcus spp. isolated from the food industry and nucleotide sequence of the resistance plasmid pST827

K.A. ALBASHERI AND W.J. MITCHELL. 1995. Maltose metabolism in the obligate anaerobe Clostridium acetobutylicum was studied. The sugar is accumulated via an energy-dependent transport process which is not a phosphotransferase. Cell extracts were incapable of phosphorylating maltose in the presence or absence of phosphoenolpyruvate or ATP, but exhibited hydrolytic activity against a range of glucoside substrates. The activity was predominantly in the soluble fraction of cell extracts, indicating a cytoplasmic location in the cell. Gel filtration on Sephadex G100 indicated the presence of at least two α-glucosidases. One enzyme (maltase) was active with maltose and maltotriose, while the other (pNPGase) hydrolysed isomaltose and several glucoside analogues, but neither showed activity against starch. Both glucosidases were induced by isomaltose, maltose, glucose and starch, but not by xylose, sucrose or cellobiose. In the presence of both glucose and maltose, growing cells showed a preference for glucose, apparently due to regulation of maltose transport, which did not occur in glucose-grown cells.     

Purification and characterization of a new type of alpha-glucosidase from Paecilomyces lilacinus that has transglucosylation activity to produce alpha-1,3- and alpha-1,2-linked oligosaccharides

A fungus producing an alpha-glucosidase that synthesizes alpha-1,3- and alpha-1,2-linked glucooligosaccharides by transglucosylation was isolated and identified as Paecilomyces lilacinus. The cell-bound enzyme responsible for the synthesis was extracted by suspension of mycelia with 0.1 M phosphate buffer (pH 8.0), and the extract was purified. The molecular weight and the isoelectric point were estimated to be 54,000 and 9.1, respectively. The enzyme was most active at pH 5.0 and 65 degres C. The enzyme hydrolyzed maltose, nigerose, and kojibiose. The enzyme also hydrolyzed soluble starch and amylose with the rate toward maltose. p-Nitro-phenyl alpha-glucoside and isomaltose were not good substrates. The enzyme had high transglucosylation activity to synthesize oligosaccharides containing alpha-1,3- and alpha-1,2-linkages. At an early stage of the reaction, considerable maltotriose, 4-O-alpha-nigerosyl-D-glucose, and 4-O-alpha-kojibiosyl-D-glucose were synthesized. Afterwards, nigerose and kojibiose were accumulated gradually with glucose as an acceptor.     

Transglucosylation activities of multiple forms of alpha-glucosidase from spinach

Transglucosylation activities of spinach alpha-glucosidase I and IV, which have different substrate specificity for hydrolyzing activity, were investigated. In a maltose mixture, alpha-glucosidase I, which has high activity toward not only maltooligosaccharides but also soluble starch and can hydrolyze isomaltose, produced maltotriose, isomaltose, and panose, and alpha-glucosidase IV, which has high activity toward maltooligosaccharides but faint activity toward soluble starch and isomaltose, produced maltotriose, kojibiose, and 2,4-di-alpha-D-glucosyl-glucose. Transglucosylation to sucrose by alpha-glucosidase I and IV resulted in the production of theanderose and erlose, respectively, showing that spinach alpha-glucosidase I and IV are useful to synthesize the alpha-1,6-glucosylated and alpha-1,2- and 1,4-glucosylated products, respectively.     

Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme.

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-alpha,alpha-1,1-glucose) and maltose (glucosyl-alpha1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of approximately 68 kDa. However, active enzyme exhibited a molecular mass of approximately 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42-45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in >or= 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8-10% free glucose. The K(m) for maltose was approximately 10 mm, whereas for trehalose it was approximately 90 mm. While beta,beta-trehalose, isomaltose (alpha1,6-glucose disaccharide), kojibiose (alpha1,2) or cellobiose (beta1,4) were not substrates for TreS, nigerose (alpha1,3-glucose disaccharide) and alpha,beta-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [(3)H]Trehalose is converted to [(3)H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [(14)C]maltose produces [(14)C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as [(3)H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of [(3)H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry.     

Molecular determinants of substrate recognition in thermostable alpha-glucosidases belonging to glycoside hydrolase family 13

Bacillus stearothermophilus alpha-1,4-glucosidase (BS) is highly specific for alpha-1,4-glucosidic bonds of maltose, maltooligosaccharides and alpha-glucans. Bacillus thermoglucosdasius oligo-1,6-glucosidase (BT) can specifically hydrolyse alpha-1,6 bonds of isomaltose, isomaltooligosaccharides and alpha-limit dextrin. The two enzymes have high homology in primary structure and belong to glycoside hydrolase family 13, which contain four conservative regions (I, II, III and IV). The two enzymes are suggested to be very close in structure, even though there are strict differences in their substrate specificities. Molecular determinants of substrate recognition in these two enzymes were analysed by site-directed mutagenesis. Twenty BT-based mutants and three BS-based mutants were constructed and characterized. Double substitutions in BT of Val200 -->Ala in region II and Pro258 -->Asn in region III caused an appearance of maltase activity compared with BS, and a large reduction of isomaltase activity. The values of k(0)/K(m) (s(-1). mM(-1)) of the BT-mutant for maltose and isomaltose were 69.0 and 15.4, respectively. We conclude that the Val/Ala200 and Pro/Asn258 residues in the alpha-glucosidases may be largely responsible for substrate recognition, although the regions I and IV also exert a slight influence. Additionally, BT V200A and V200A/P258N possessed high hydrolase activity towards sucrose.     

Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan.

The action of neopullulanase from Bacillus stearothermophilus on many oligosaccharides was tested. The enzyme hydrolyzed not only alpha-(1----4)-glucosidic linkages but also specific alpha-(1----6)-glucosidic linkages of several branched oligosaccharides. When pullulan was used as a substrate, panose, maltose, and glucose, in that order, were produced as final products at a final molar ratio of 3:1:1. According to these results, we proposed a model for the pattern of action of neopullulanase on pullulan as follows. In the first step, the enzyme hydrolyzes only alpha-(1----4)-glucosidic linkages on the nonreducing side of alpha-(1----6) linkages of pullulan and produces panose and several intermediate products composed of some panose units. In the second step, taking 6(2)-O-alpha-(6(3)-O-alpha-glucosyl-maltotriosyl)-maltose as an example of one of the intermediate products, the enzyme hydrolyzes either alpha-(1----4) (the same position as that described above) or alpha-(1----6) linkages and produces panose or 6(3)-O-alpha-glucosyl-maltotriose plus maltose, respectively. In the third step, the alpha-(1----4) linkage of 6(3)-O-alpha-glucosyl-maltotriose is hydrolyzed by the enzyme, and glucose and another panose are produced. To confirm the model of the pattern of action, we extracted intermediate products produced from pullulan by neopullulanase and analyzed the structures by glucoamylase, pullulanase, and neopullulanase analyses. The experimental results supported the above-mentioned model of the pattern of action of neopullulanase on pullulan.     

Val216 decides the substrate specificity of alpha-glucosidase in Saccharomyces cerevisiae.

Differences in the substrate specificity of alpha-glucosidases should be due to the differences in the substrate binding and the catalytic domains of the enzymes. To elucidate such differences of enzymes hydrolyzing alpha-1,4- and alpha-1,6-glucosidic linkages, two alpha-glucosidases, maltase and isomaltase, from Saccharomyces cerevisiae were cloned and analyzed. The cloned yeast isomaltase and maltase consisted of 589 and 584 amino acid residues, respectively. There was 72.1% sequence identity with 165 amino acid alterations between the two alpha-glucosidases. These two alpha-glucosidase genes were subcloned into the pKP1500 expression vector and expressed in Escherichia coli. The purified alpha-glucosidases showed the same substrate specificities as those of their parent native glucosidases. Chimeric enzymes constructed from isomaltase by exchanging with maltase fragments were characterized by their substrate specificities. When the consensus region II, which is one of the four regions conserved in family 13 (alpha-amylase family), is replaced with the maltase type, the chimeric enzymes alter to hydrolyze maltose. Three amino acid residues in consensus region II were different in the two alpha-glucosidases. Thus, we modified Val216, Gly217, and Ser218 of isomaltase to the maltase-type amino acids by site-directed mutagenesis. The Val216 mutant was altered to hydrolyze both maltose and isomaltose but neither the Gly217 nor the Ser218 mutant changed their substrate specificity, indicating that Val216 is an important residue discriminating the alpha-1,4- and 1,6-glucosidic linkages of substrates.     

Comparison of the action of glucoamylase and glucosyltransferase on D-glucose, maltose, and malto-oligosaccharides.

The action patterns of glucoamylase (amyloglucosidase) and glucosyltransferase (transglucosylase) on D-[1-14C]glucose, [1-14C]maltose, and [1-14C]malto-oligosaccharides (labeled at position 1 of the D-glucose group at the reducing end) have been investigated by paper-chromatographic and oligosaccharide-mapping techniques. Under the conditions of the experiments, the extent of conversion of D-glucose and of maltose into new oligosaccharides was 2.2 and 1.9% with glucoamylase, and 5.7 and 33% with glucosyltransferase. The major oligosaccharides produced by both enzymes were isomaltose (6-O-alpha-D-glucopyranosyl-alpha-D-glucose), panose (O-alpha-D-glucopyranosyl (1 leads to 6)-O-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-glucose), and nigerose (3-O-alpha-D-glucopyranosyl-alpha-D-glucose). The glucosyltransferase also synthesized oligosaccharides from malto-oligosaccharides of higher molecular weight to yield compounds having alpha-(1 leads to 6)-linked D-glucosyl groups at the non-reducing ends. Glucoamylase exhibited little, if any, such activity on malto-oligosaccharides.     

Glycogenolytic enzymes in sporulating yeast.

During meiosis in Saccharomyces cerevisiae, the polysaccharide glycogen is first synthesized and then degraded during the period of spore maturation. We have detected, in sporulating yeast strains, an enzyme activity which is responsible for the glycogen catabolism. The activity was absent in vegetative cells, appeared coincidently with the beginning of glycogenolysis and the appearance of mature ascospores, and increased progressively until spourlation was complete. The specific activity of glycogenolytic enzymes in the intact ascus was about threefold higher than in isolated spores. The glycogenolysis was not due to combinations of phosphorylase plus phosphatase or amylase plus maltase. Nonsporulating cells exhibited litle or no glycogen catabolism and contained only traces of glycogenolytic enzyme, suggesting that the activity is sporulation specific. The partially purified enzyme preparation degraded amylose and glycogen, releasing glucose as the only low-molecular-weight product. Maltotriose was rapidly hydrolyzed; maltose was less susceptible. Alpha-methyl-D-glucoside, isomaltose, and linear alpha-1,6-linked dextran were not attacked. However, the enzyme hydrolyzed alpha-1,6-glucosyl-Schardinger dextrin and increased the beta-amylolysis of beta-amylase-limit dextrin. Thus, the preparation contains alpha-1,4- and alpha-1,6-glucosidase activities. Sephadex G-150 chromatography partially resolved the enzyme into two activities, one of which may be a glucamylase and the other a debranching enzyme.     

Stopped-flow fluorescence and steady-state kinetic studies of ligand-binding reactions of glucoamylase from Aspergillus niger.

The presteady-state and steady-state kinetics of the binding and hydrolysis of substrates, maltose and isomaltose, and the transition-state analogue, gluconolactone, by glucoamylase from Aspergillus niger were investigated using initial-rate, stopped-flow and steady-state methods. The change in the intrinsic fluorescence of the enzyme was monitored. Distinct mechanistic differences were observed in the interaction of the enzyme with maltose compared to isomaltose. Hydrolysis of maltose requires a three-step mechanism, whereas that of isomaltose involves at least one additional step. The rates of an observed conformational change, which is the second discernible step of the reactions, clearly show a tighter binding of maltose compared to isomaltose, probably because the reverse rate constants differ. Compared to the non-enzymic hydrolysis the transition-state stabilization energy of glucoamylase is approximately -66 kJ/mol with maltose and only -14 kJ/mol with isomaltose. Kinetic analysis of the binding of the inhibitor, gluconolactone, implies that independent interactions of two molecules occur. One of these, apparently, is a simple, fast association reaction in which gluconolactone is weakly bound. The other resembles binding of maltose, involving a fast association followed by a conformational change. Based on the results obtained, we propose new reaction mechanisms for Aspergillus glucoamylase.