Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis.

Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments indicated that separate phosphotransferase systems (enzymes II) existed for glucose, maltose, and sucrose. [14C]maltose transport was inhibited by excess (10 mM) glucose and to a lesser extent by sucrose (90 and 46%, respectively). [14C]glucose and [14C]sucrose transports were not inhibited by an excess of maltose. Since [14C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of Pi was increased from 0 to 100 mM, a maltose phosphorylase (Km for Pi, 9.5 mM) was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for alpha-glucose 1-phosphate (Km, 0.8 mM). Only sucrose-grown cells possessed sucrose hydrolase activity (Km, 3.1 mM), and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities.     

Uptake and metabolism of sucrose by Streptococcus lactis

Transport and metabolism of sucrose in Streptococcus lactis K1 have been examined. Starved cells of S. lactis K1 grown previously on sucrose accumulated [14C]sucrose by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) (sucrose-PTS; Km, 22 microM; Vmax, 191 mumol transported min-1 g of dry weight of cells-1). The product of group translocation was sucrose 6-phosphate (6-O-phosphoryl-D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside). A specific sucrose 6-phosphate hydrolase was identified which cleaved the disaccharide phosphate (Km, 0.10 mM) to glucose 6-phosphate and fructose. The enzyme did not cleave sucrose 6'-phosphate(D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside-6'-phosphate). Extracts prepared from sucrose-grown cells also contained an ATP-dependent mannofructokinase which catalyzed the conversion of fructose to fructose 6-phosphate (Km, 0.33 mM). The sucrose-PTS and sucrose 6-phosphate hydrolase activities were coordinately induced during growth on sucrose. Mannofructokinase appeared to be regulated independently of the sucrose-PTS and sucrose 6-phosphate hydrolase, since expression also occurred when S. lactis K1 was grown on non-PTS sugars. Expression of the mannofructokinase may be negatively regulated by a component (or a derivative) of the PTS.     

Plasmid-mediated uptake and metabolism of sucrose by Escherichia coli K-12.

The conjugative plasmid pUR400 determines tetracycline resistance and enables cells of Escherichia coli K-12 to utilize sucrose as the sole carbon source. Three types of mutants affecting sucrose metabolism were derived from pUR400. One type lacked a specific transport system (srcA); another lacked sucrose-6-phosphate hydrolase (scrB); and the third, a regulatory mutant, expressed both of these functions constitutively (scrR). In a strain harboring pUR400, both transport and sucrose-6-phosphate hydrolase were inducible by fructose, sucrose, and raffinose; if a scrB mutant was used, fructose was the only inducer. These data suggested that fructose or a derivative acted as an endogenous inducer. Sucrose transport and sucrose-6-phosphate hydrolase were subject to catabolite repression; these two functions were not expressed in an E. coli host (of pUR400) deficient in the adenosine 3-,5'-phosphate receptor protein. Sucrose uptake (apparent Km = 10 microM) was dependent on the scrA gene product and on the phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) of the host. The product of sucrose uptake (via group translocation) was identified as sucrose-6-phosphate, phosphorylated at C6 of the glucose moiety. Intracellular sucrose-6-phosphate hydrolase catalyzed the hydrolysis of sucrose-6-phosphate (Km = 0.17 mM), sucrose (Km = 60 mM), and raffinose (Km = 150 mM). The active enzyme was shown to be a dimer of Mr 110,000.     

Purification and properties of Cellvibrio gilvus cellobiose phosphorylase

The cellobiose phosphorylase (EC 2.4.1.20) of Cellvibrio gilvus, which is an endocellular enzyme, has been purified 196-fold with a recovery of 11% and a specific activity of 27.4 mumol of glucose 1-phosphate formed/min per mg of protein. The purification procedure includes fractionation with protamine sulphate, and hydroxyapatite and DEAE-Sephadex A-50 chromatography. The enzyme appears homogeneous on polyacrylamide-gel electrophoresis, and a molecular weight of 280 000 was determined by molecular-sieve chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a single band and mol.wt. 72 000, indicating that cellobiose phosphorylase consists of four subunits. The enzyme had a specificity for cellobiose, requiring Pi and Mg2+ for phosphorylation, but not for cellodextrin, gentibiose, laminaribiose, lactose, maltose, kojibiose and sucrose. The enzyme showed low thermostability, an optimum pH of 7.6 and a high stability in the presence of 2-mercaptoethanol or dithiothreitol. The Km values for cellobiose and Pi were 1.25 mM and 0.77 mM respectively. Nojirimycin acted as a powerful pure competitive inhibitor (with respect to cellobiose) of the enzyme (Ki = 45 microM). Addition of thiol-blocking agents to the enzyme caused 56% inhibition at 500 microM-N-ethylmaleimide and 100% at 20 microM-p-chloromercuribenzoate.     

Phosphorylation and metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae

Not only sucrose but the five isomeric alpha-D-glucosyl-D-fructoses trehalulose, turanose, maltulose, leucrose, and palatinose are utilized by Klebsiella pneumoniae as energy sources for growth, thereby undergoing phosphorylation by a phosphoenolpyruvate-dependent phosphotransferase system uniformly at 0-6 of the glucosyl moiety. Similarly, maltose, isomaltose, and maltitol, when exposed to these conditions, are phosphorylated regiospecifically at O-6 of their non-reducing glucose portion. The structures of these novel compounds have been established unequivocally by enzymatic analysis, acid hydrolysis, FAB negative-ion spectrometry, and 1H and 13C NMR spectroscopy. In cells of K. pneumoniae, hydrolysis of sucrose 6-phosphate is catalyzed by sucrose 6-phosphate hydrolase from Family 32 of the glycosylhydrolase superfamily. The five 6'-O-phosphorylated alpha-D-glucosyl-fructoses are hydrolyzed by an inducible (approximately 49-50 Kda) phospho-alpha-glucosidase from Family 4 of the glycosylhydrolase superfamily.     

The uptake and metabolism of glucose, maltose and starch by the rumen ciliate Epidinium ecaudatum caudatum.

[14C]Glucose taken up by Epidinium ecaudatum caudatum was found in the pool, in the protozoal polysaccharide and in the bacteria associated with the protozoa. The amount incorporated into the polysaccharide depended on the square of the glucose concentration. Evidence was obtained that glucose was probably taken up initially into the pool unchanged, and then rapidly converted into glucose 6-phosphate and maltose which were subsequently hydrolysed to glucose. [14C]-Maltose was taken up at 20 to 30% of the rate of [14C]glucose, with 14C appearing initially in maltose and glucose 6-phosphate. 14C from 14C-labelled soluble starch appeared in the pool as maltose, glucose 6-phosphate and glucose in that order, but incorporation into protozoal polysaccaride was poor. Hexokinase, phosphoglucomutase, alpha-glucan and maltose phosphorylases, glucose 6-phosphatase and maltase activities were found in the protozoa.     

Transport and metabolism of trehalose in Escherichia coli and Salmonella typhimurium

The metabolism of trehalose in wild type cells of Escherichia coli and Salmonella typhimurium has been investigated. Intact cells of Escherichia coli (grown on trehalose) accumulated [¹⁴C]-trehalose as [¹⁴C]-trehalose 6-phosphate. Toluene-treated cells catalyzed the synthesis of the [¹⁴C]-sugar phosphate from [¹⁴C]-trehalose and phosphoenolpyruvate; ATP did not serve as phosphoryl donor. Trehalose 6-phosphate could subsequently be hydrolyzed by trehalose 6-phosphate hydrolase, an enzyme which catalyzes the hydrolysis of the disaccharide phosphate into glucose and glucose 6-phosphate. Both Escherichia coli and Salmonella typhimurium induced this enzyme when they grew on trehalose.These findings suggest that trehalose is transported in these bacteria by an inducible phosphoenolpyruvate:trehalose phosphotransferase system.The presence of a constitutive trehalase was also detected.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanosulfonic acid - PEP phosphoenolpyruvate - PTS phosphoenolpyruvate: glycose phosphotransferase system - O.D. optical density     

Sub-compartmentation of the 'cytosolic' glucose 6-phosphate pool in cultured rat hepatocytes

[14C]Glucose release either from endogenous 14C-prelabelled glycogen or from added 14C-labelled glucose 6-phosphate was measured in filipin-treated, permeabilized hepatocytes in 48 h culture. [14C]Glucose output from prelabelled glycogen was not altered by the addition of 5 mM glucose 6-phosphate to the incubation medium. Conversely, [14C]glucose release from 5 mM labelled glucose 6-phosphate was not influenced by different glycogen concentrations in the cells. Moreover, in the permeabilized cells the anion transport inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) inhibited only the liberation of [14C]glucose from labelled glucose 6-phosphate but not from glycogen. It is therefore concluded that there exist at least 2 separate, mutually non-accessible glucose 6-phosphate pools in cultured rat hepatocytes, one linked to glycogenolysis and the other to gluconeogenesis.     

Phosphoenolpyruvate-dependent maltose:phosphotransferase activity in Fusobacterium mortiferum ATCC 25557: specificity, inducibility, and product analysis.

Phosphoenolypyruvate-dependent maltose:phosphotransferase activity was induced in cells of Fusobacterium mortiferum ATCC 25557 during growth on maltose. The disaccharide was rapidly metabolized by washed cells maintained under anaerobic conditions, but fermentation ceased immediately upon exposure of the cell suspension to air. Coincidentally, high levels of a phosphorylated derivative accumulated within the cells. Chemical and enzymatic analyses, in conjunction with data from 1H, 13C, and 31P nuclear magnetic resonance spectroscopy, established the structure of the purified compound as 6-O-phosphoryl-alpha-D-glucopyranosyl-(1-4)-D-glucose (maltose 6-phosphate). A method for the preparation of substrate amounts of this commercially unavailable disaccharide phosphate is described. Permeabilized cells of F. mortiferum catalyzed the phosphoenolpyruvate-dependent phosphorylation of maltose under aerobic conditions. However, the hydrolysis of maltose 6-phosphate (to glucose 6-phosphate and glucose) by permeabilized cells or cell-free preparations required either an anaerobic environment or addition of dithiothreitol to aerobic reaction mixtures. The first step in dissimilation of the phosphorylated disaccharide appears to be catalyzed by an oxygen-sensitive maltose 6-phosphate hydrolase. Cells of F. mortiferum, grown previously on maltose, fermented a variety of alpha-linked glucosides, including maltose, turanose, palatinose, maltitol, alpha-methylglucoside, trehalose, and isomaltose. Conversely, cells grown on the separate alpha-glucosides also metabolized maltose. For this anaerobic pathogen, we suggest that the maltose:phosphotransferase and maltose 6-phosphate hydrolase catalyze the phosphorylative translocation and cleavage not only of maltose but also of structurally analogous alpha-linked glucosides.     

Lactose metabolism in Streptococcus lactis: phosphorylation of galactose and glucose moieties in vivo.

Starved cells of Streptococcus lactis ML3 grown previously on lactose, galactose, or maltose were devoid of adenosine 5'-triphosphate contained only three glycolytic intermediates: 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate (PEP). The three metabolites (total concentration, ca 40 mM) served as the intracellular PEP potential for sugar transport via PEP-dependent phosphotransferase systems. When accumulation of [14C]lactose by iodoacetate-inhibited starved cells was abolished within 1 s of commencement of transport, a phosphorylated disaccharide was identified by autoradiography. The compound was isolated by ion-exchange (borate) chromatography, and enzymatic analysis showed that the derivative was 6-phosphoryl-O-beta-D-galactopyranosyl (1 leads to 4')-alpha-D-glucopyranose (lactose 6-phosphate). After maximum lactose uptake (ca. 15 mM in 15 s) the cells were collected by membrane filtration and extracted with trichloroacetic acid. Neither free nor phosphorylated lactose was detected in cell extracts, but enzymatic analysis revealed high levels of galactose 6-phosphate and glucose 6-phosphate. The starved organisms rapidly accumulated glucose, 2-deoxy-D-glucose, methyl-beta-D-thiogalactopyranoside, and o-nitrophenyl-beta-D-galactopyranoside in phosphorylated form to intracellular concentrations of 32, 32, 42, and 38.5 mM, respectively. In contrast, maximum accumulation of lactose (ca. 15 mM) was only 40 to 50% that of the monosaccharides. From the stoichiometry of PEP-dependent lactose transport and the results of enzymatic analysis, it was concluded that (i) ca. 60% of the PEP potential was utilized via the lactose phosphotransferase system for phosphorylation of the galactosyl moiety of the disaccharide, and (ii) the residual potential (ca. 40%) was consumed during phosphorylation of the glucose moiety.     

Trehalose Metabolism by Bacillus popilliae

Trehalose was found to be utilized more readily than glucose for the growth of Bacillus popilliae NRRL B-2309MC. The pathway of degradation of trehalose was elucidated and found to differ from that reported for other organisms. Trehalase and trehalose phosphorylase activities could not be detected. Rather, trehalose was found to undergo phosphoenolpyruvate (PEP)-dependent phosphorylation, and the resulting trehalose 6-phosphate was cleaved by a phosphotrehalase to equimolar amounts of glucose and glucose 6-phosphate. The phosphotrehalase was purified 34-fold and shown to have a pH optimum of 6.5 to 7.0 and a K(m) for trehalose 6-phosphate of 1.8 mM. A mutant missing the phosphotrehalase failed to grow on trehalose but grew normally on other sugars. The mutant accumulated [(14)C]trehalose as [(14)C]trehalose 6-phosphate. Phosphorylation of trehalose by dialyzed extracts was at least 25 times faster with PEP than with adenosine 5'-triphosphate, and the phosphorylation activity was associated primarily with the particulate fraction. These data and the results of studies of [(14)C]trehalose uptake suggest that trehalose is transported into the cell as trehalose 6-phosphate by a PEP:sugar phosphotransferase system. Cell extracts of other strains of B. popilliae were also found to produce [(14)C]sugar phosphate from [(14)C]trehalose and to have phosphotrehalase activity.     

Glycogen synthesis in the fungus Neurospora crassa.

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [14C]glucose from ADP-[14C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose : glycogen glucosyltransferase as compared with those of the already known UDP glucose : glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-14C]glucose or ADP-[14C]glucose released all the radioactivity as maltose after alpha or beta amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[14C]glucose was the substrate but the stimulation was much greater with ADP-[14C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent &on the presence of a 'primer' of the alpha 1 leads to 4 glucan type.     

Characterization of a maltose transport system in Clostridium acetobutylicum ATCC 824

The utilization of maltose by Clostridium acetobutylicum ATCC 824 was investigated. Glucose was used preferentially to maltose, when both substrates were present in the medium. Maltose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity was detected in extracts prepared from cultures grown on maltose, but not glucose or sucrose, as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that the specificity for maltose is contained entirely within the membrane in this organism. A putative gene system for the maltose PTS was identified (from the C. acetobutylicum ATCC 824 genome sequence), encoding an enzyme II^Mal and a maltose 6-phosphate hydrolase. Journal of Industrial Microbiology & Biotechnology (2001) 27, 298–306. Received 12 September 2000/ Accepted in revised form 30 November 2000     

Inducible phosphoenolpyruvate-dependent hexose phosphotransferase activities in Escherichia coli

1. A method is described for measuring the rate of phosphoenolpyruvate-dependent phosphotransferase activity for a variety of hexoses in toluene-treated suspensions of Escherichia coli. 2. The specific activities of the phosphotransferases that catalyse the phosphorylation of hexoses are greatly affected by the carbon source for growth. 3. In all strains of E. coli tested, fructose phosphotransferase activity is induced by growth on fructose. 4. Strains of E. coli differ greatly in the rate at which they phosphorylate glucose, but all strains possess at least a low glucose phosphotransferase activity under any tested condition of growth. Glucose phosphotransferase activity is further induced by growth on glucose; this does not occur in a mutant that lacks the ability to take up methyl alpha-d-[(14)C]glucopyranoside and hence grows poorly on glucose. 5. When growing on fructose, two strains of E. coli synthesize the inducible glucose phosphotransferase system gratuitously, and to specific activities higher than observed during growth on glucose. A phosphotransferase catalysing the phosphorylation of mannose is similarly induced.     

Glucose metabolism in mouse pancreatic islets

1. Rates of glucose oxidation, lactate output and the intracellular concentration of glucose 6-phosphate were measured in mouse pancreatic islets incubated in vitro. 2. Glucose oxidation rate, measured as the formation of (14)CO(2) from [U-(14)C]glucose, was markedly dependent on extracellular glucose concentration. It was especially sensitive to glucose concentrations between 1 and 2mg/ml. Glucose oxidation was inhibited by mannoheptulose and glucosamine but not by phlorrhizin, 2-deoxyglucose or N-acetylglucosamine. Glucose oxidation was slightly stimulated by tolbutamide but was not significantly affected by adrenaline, diazoxide or absence of Ca(2+) (all of which may inhibit glucose-stimulated insulin release), by arginine or glucagon (which may stimulate insulin release) or by cycloheximide (which may inhibit insulin synthesis). 3. Rates of lactate formation were dependent on the extracellular glucose concentration and were decreased by glucosamine though not by mannoheptulose; tolbutamide increased the rate of lactate output. 4. Islet glucose 6-phosphate concentration was also markedly dependent on extracellular glucose concentration and was diminished by mannoheptulose or glucosamine; tolbutamide and glucagon were without significant effect. Mannose increased islet fructose 6-phosphate concentration but had little effect on islet glucose 6-phosphate concentration. Fructose increased islet glucose 6-phosphate concentration but to a much smaller extent than did glucose. 5. [1-(14)C]Mannose and [U-(14)C]fructose were also oxidized by islets but less rapidly than glucose. Conversion of [1-(14)C]mannose into [1-(14)C]glucose 6-phosphate or [1-(14)C]glucose could not be detected. It is concluded that metabolism of mannose is associated with poor equilibration between fructose 6-phosphate and glucose 6-phosphate. 6. These results are consistent with the idea that glucose utilization in mouse islets may be limited by the rate of glucose phosphorylation, that mannoheptulose and glucosamine may inhibit glucose phosphorylation and that effects of glucose on insulin release may be mediated through metabolism of the sugar.     

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-1?C]glucose 1-phosphate, [U-1?C]sucrose, [U-1?C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-1?C]sucrose plus unlabelled equimolar glucose 1-phosphate. C1?-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced 1?C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-1?C]glucose 1-phosphate or adenosine-[U-1?C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C1?C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.     

Glycogen synthesis in the fungus Neurospora crassa

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [¹⁴C]glucose from ADP-[¹⁴C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose: glycogen glucosyltransferase as compared with those of the already known UDP glucose: glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-[¹⁴C]glucose or ADP-[¹⁴C]glucose released all the radioactivity as maltose after α or β amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[¹⁴C]glucose was the substrate but the stimulation was much greater with ADP-[¹⁴C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent on the presence of a ‘primer’ of the α 1 → 4 glucan type.     

Factors Affecting Glucose and Maltose Phosphorylation by the Ruminal Bacterium Megasphaera elsdenii

The objectives of this study were to examine the effects of growth substrate and extracellular pH on phosphoenolpyruvate-dependent glucose phosphorylation as well as to examine how maltose is phosphorylated by the ruminal bacterium Megasphaera elsdenii B159. Phosphoenolpyruvate-dependent glucose phosphorylation by toluene-treated cells was constitutive, and glucose phosphorylation was reduced by 69% at pH 5.0. When toluene-treated cells were incubated in histidine buffer, little maltose phosphorylation occurred in the absence of inorganic phosphate. However, the addition of increasing concentrations of either potassium or sodium phosphate increased maltose phosphorylation. Maximal phosphorylation activity was observed at between 25 and 50 mM of either inorganic phosphate source. Compared with the control incubations, maltose phosphorylation was increased over threefold with 25 mM of either potassium or sodium phosphate. Phosphoglucomutase activity was detected in cell extracts of M. elsdenii B159, and this enzyme had a K _m of 3.2 mM for glucose-1-P and a V _max of 1836 nmol of NADP⁺ reduced/mg of protein per min. Maltose was also hydrolyzed by an inducible maltase (K _m , 1.19 mM). To our knowledge, this is the first report of a maltose phosphorylase and a maltase in M. elsdenii. Received: 3 November 1999 / Accepted: 5 January 2000     

Kinetic characterization of rabbit skeletal muscle phosphorylase ab hybrid

Phosphorylase ab was prepared in vitro by partial phosphorylation of rabbit skeletal muscle phosphorylase b and was isolated by DEAE-Sephacel chromatography. Its phosphorylated and non-phosphorylated subunits could not be distinguished by different affinity to substrates, activators or inhibitors, indicating their coordinated function. In the absence of nucleotide activators, the Km values for Pi and glucose-1-P were 28 mM and 18 mM, respectively. Activity in the presence of 16 mM glucose-1-P was doubled by 10(-4) M AMP or 10(-3) M IMP, mainly by lowering the Km for glucose-1-P. Half-maximum activation was exerted by 2 microM AMP or 0.1 mM IMP. Activation by these nucleotides showed no cooperativity. Glucose exerted competitive inhibition with respect to glucose-1-P, while for the inhibition by glucose-6-P an allosteric mechanism is suggested; the appropriate Ki values were 4.5 mM and 1.5 mM, respectively. The Hill coefficient for glucose-1-P binding was about 1.0, even in the presence of glucose (up to 10 mM), but 10 mM glucose-6-P lowered it to 0.47, indicating a negative heterotropic cooperativity. Effective regulation of the activity of phosphorylase ab by physiological concentrations of Pi, AMP, IMP and glucose-6-P suggests its metabolic control under in vivo condition.     

6-phospho-alpha-D-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases.

The Fusobacterium mortiferum malH gene, encoding 6-phospho-alpha-glucosidase (maltose 6-phosphate hydrolase; EC 3.2.1.122), has been isolated, characterized, and expressed in Escherichia coli. The relative molecular weight of the polypeptide encoded by malH (441 residues; Mr of 49,718) was in agreement with the estimated value (approximately 49,000) obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme purified from F. mortiferum. The N-terminal sequence of the MalH protein obtained by Edman degradation corresponded to the first 32 amino acids deduced from the malH sequence. The enzyme produced by the strain carrying the cloned malH gene cleaved [U-14C]maltose 6-phosphate to glucose 6-phosphate (Glc6P) and glucose. The substrate analogs p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alphaGlc6P) and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate (4MU alphaGlc6P) were hydrolyzed to yield Glc6P and the yellow p-nitrophenolate and fluorescent 4-methylumbelliferyl aglycons, respectively. The 6-phospho-alpha-glucosidase expressed in E. coli (like the enzyme purified from F. mortiferum) required Fe2+, Mn2+, Co2+, or Ni2+ for activity and was inhibited in air. Synthesis of maltose 6-phosphate hydrolase from the cloned malH gene in E. coli was modulated by addition of various sugars to the growth medium. Computer-based analyses of MalH and its homologs revealed that the phospho-alpha-glucosidase from F. mortiferum belongs to the seven-member family 4 of the glycosylhydrolase superfamily. The cloned 2.2-kb Sau3AI DNA fragment from F. mortiferum contained a second partial open reading frame of 83 residues (designated malB) that was located immediately upstream of malH. The high degree of sequence identity of MalB with IIB(Glc)-like proteins of the phosphoenol pyruvate dependent:sugar phosphotransferase system suggests participation of MalB in translocation of maltose and related alpha-glucosides in F. mortiferum.