Km For Glucose Research Articles

A Mutation Ser213/Asn in the Hexokinase 1 fromSchizosaccharomyces pombeIncreases Its Affinity for Glucose

Alignment of amino acids of the region implicated in glucose binding from a series of hexokinases showed thatSchizosaccharomyces pombehexokinase 1 had a Ser residue in a place where all other kinases had an Asn. We changed an AGT codon to AAT to place an Asn in the Ser213position. This mutation decreased Kmfor glucose from 9.4 mM to 1.6 mM and the ratio Vmax(Fructose)/Vmax(Glucose) from 5 to 2.5. Also the Kmfor 2-deoxyglucose decreased from 2.7 mM to 0.8 mM. A mutation in the similar position ofS. pombehexokinase 2 (Asn196/Ser) increased the Kmfor glucose from 0.16 mM to 0.56 mM. Fermentation of glucose is not detectable in aS. pombemutant with only hexokinase 1 activity but expression of thehxk1S213/Ngene conferred ability to ferment the sugar. While the mutated hexokinase 1 partially mimickedS. cerevisiaehexokinase II in catabolite repression of invertase, the wild type one could not substitute for it.

Glucose transport in a methylotrophic yeast Hansenula polymorpha.

Glucose transport was studied in a methylotrophic yeast Hansenula polymorpha. Two kinetically different glucose transport systems were revealed in cells grown under different growth conditions. Glucose-repressed cells exhibited a low-affinity transport system (Km for glucose 1.75 mM) while glucose-derepressed and ethanol-grown cells had a high-affinity transport system (Km for glucose 0.05-0.06 mM). The high- and low-affinity transport systems differed in substrate specificity, sensitivity to pH, dinitrophenol and protonophore carbonyl cyanide-m-chlorophenyl-hydrazone. The kinetic rearrangement of the glucose transport system in response to altered growth conditions was dependent on de novo protein synthesis.

Intracerebroventricular Administration of Antisense Oligodeoxynucleotide against GLUT2 Glucose Transporter mRNA Reduces Food Intake, Body Weight Change and Glucoprivic Feeding Response in Rats ,

The GLUT2 glucose transporter, which may play a glucose-sensing role in hepatocyte and islet beta cells because of its low affinity and high Km for glucose, has been identified in some discrete brain areas that are related to feeding behavior and energy metabolism. We tested the hypothesis that brain GLUT2 may play a role in the control of food intake by antisense technology loss-of-function analysis. Antisense oligonucleotides directed against GLUT2 mRNA were administered intracerebroventricularly to eight rats daily for 13 days. Another eight rats were administered intracerebroventricularly with missense oligonucleotides as the control. Food intake was monitored by a computerized feeding system. Data were analyzed using a one-way general linear model or Mann-Whitney U test when appropriate. Cumulative food intake and body weight change in antisense-treated rats were significantly lower (18 and 160%, respectively) in the group treated with antisense oligonucleotides than in the group treated with missense control oligonucleotides. There was no increase in cumulative food intake in response to 2-deoxyglucose challenge in rats treated with antisense oligonucleotide, but in those treated with missense control oligonucleotide, cumulative food intake was fivefold greater in response to 2-deoxyglucose. These data suggest a possible role of brain GLUT2 in the regulation of food intake and body energy stores.

Changes in pancreatic islet glucokinase and hexokinase activities with increasing age, obesity, and the onset of diabetes.

We examined changes in high- and low-Km glucose phosphorylating activity in pancreatic islet extracts from the prediabetic Zucker diabetic fatty (ZDF) rat between 5-6 weeks and 12 weeks of age (after the onset of diabetes). Comparisons were made between the activity observed in the ZDF rat and that seen in the ZDF lean control (ZLC) rat and the obese nondiabetic Zucker fatty (ZF) rat. At 5-6 weeks of age, insulin resistant ZDF and ZF rats were hyperinsulinemic, compared with the ZLC rat, but had normal plasma glucose levels. Kinetic parameters (Vmax and Km for glucose) of hexokinase (HK) and Km of glucokinase (GCK) did not differ between groups. Islet GCK activity for ZDF and ZF rats was 1.7-fold greater than in ZLC rats (P < 0.02 and P < 0.001, respectively). By 12 weeks of age, hypersecretion of insulin at 5.0 mmol/l glucose was observed in perifused islets from both obese groups relative to the ZLC rat. Islets from ZDF rats failed to increase insulin secretion in response to increased glucose concentration. Group differences in the kinetic parameters for GCK or in the Km values for HK were not significant. Islet HK activity for ZDF and ZF rats was 1.9-fold (P < 0.05) and 1.7-fold (P < 0.05) greater, respectively, than for ZLC rats. Compared with the 5- to 6-week-old animals, HK activity increased 3.1-fold (P < 0.001), 2.5-fold (P < 0.002), and 2.0-fold (P < 0.05) for ZDF, ZF, and ZLC rats, respectively. Differences in GCK activity between 5- to 6- and 12-week-old rats were not significant for any of the groups. We conclude: 1) increased islet glucose phosphorylating activity is present in insulin resistant and hyperinsulinemic ZF and ZDF rats, relative to the ZLC rat; 2) at 12 weeks of age, hyperinsulinemic ZDF and ZF rats demonstrated significant increases in HK activity, compared with lean controls; and 3) deficiency in GCK activity does not explain failure of diabetic ZDF islets to respond to glucose, since differences between diabetic ZDF and nondiabetic ZF rats were not statistically significant. Increases in pancreatic islet phosphorylating activity seem to be important in maintaining basal hyperinsulinemia in insulin-resistant animals, but do not appear to play a role in the progression to glucose intolerance and diabetes.

Coupled Glucose Transport and Metabolism in Cultured Neuronal Cells: Determination of the Rate-Limiting Step

In brain and nerves the phosphorylation of glucose, rather than its transport, is generally considered the major rate-limiting step in metabolism. Since little is known regarding the kinetic coupling between these processes in neuronal tissues, we investigated the transport and phosphorylation of [2-3H]glucose in two neuronal cell models: a stable neuroblastoma cell line (NCB20), and a primary culture of isolated rat dorsal root ganglia cells. When transport and phosphorylation were measured in series, phosphorylation was the limiting step, because intracellular glucose concentrations were the same as those outside of cells, and because the apparent Km for glucose utilization was lower than expected for the transport step. However, the apparent Km was still severalfold higher than the Km of hexokinase I. When [2-3H]glucose efflux and phosphorylation were measured from the same intracellular glucose pool in a parallel assay, rates of glucose efflux were three- to-fivefold greater than rates of phosphorylation. With the parallel assay, we observed that activation of glucose utilization by the sodium channel blocker veratridine caused a selective increase in glucose phosphorylation and was without effect on glucose transport. In contrast to results with glucose, both cell types accumulated 2-deoxy-D-[14C]glucose to concentrations severalfold greater than extracellular concentrations. We conclude from these studies that glucose utilization in neuronal cells is phosphorylation-limited, and that the coupling between transport and phosphorylation depends on the type of hexose used.

Different properties of the mitochondrial and cytosolic hexokinases in maize roots.

After tissue homogenization, 43% of the total hexokinase activity found in maize radicles was recovered in the mitochondrial fraction and 35% was soluble, in the cytosol. The maize submitochondrial particles obtained after mitochondrial sonication retained a high hexokinase activity. The mitochondrial respiration (state 4 rate) was activated by glucose. This activation was blocked by carboxyatractyloside (0.5 mM) and by oligomycin (2 micrograms/ml). The affinities for ATP and glucose of both soluble and membrane-bound maize hexokinases are similar to those of yeast hexokinase. The Km for ATP of these different forms of hexokinase varied between 0.15 and 0.37 mM, and the Km for glucose between 0.05 and 0.13 mM. A major difference between the two maize hexokinase forms is that only the mitochondrial enzyme was strongly inhibited by ADP (Ki 0.04 mM). The soluble forms of hexokinase found both in the cytosol of maize radicles and in yeast are not inhibited by ADP. In a previous report [de Meis, Grieco and Galina (1992) FEBS Lett. 308, 197-201] it was shown that the mitochondrial F1-F0-ATPase can use glucose 6-phosphate and yeast hexokinase as an ATP regenerating system. We now show that the membrane-bound hexokinase and glucose 6-phosphate can also serve as an ATP regenerating system for the mitochondria of maize radicles provided that the ADP concentration is kept below 0.05 mM. Higher ADP concentrations inhibit the reverse reaction of the mitochondrial hexokinase.

Active Site Residues of Human Brain Hexokinase as Studied by Site-specific Mutagenesis

The truncated gene of hexokinase, mini-hexokinase, starting with methionine 455 and ending at the C terminus was expressed in Escherichia coli. Mini-hexokinase lost its ability to ameliorate inhibition of glucose-6-P-inhibited mini-hexokinase in the presence of phosphate (P(i)). We suggest that the P(i) site either resides in the N-terminal half of hexokinase I or requires the N-terminal portion of the enzyme. Site-directed mutagenesis was performed to obtain two mutants of mini-hexokinase: C606S and C628S. Both are thought to be associated with the active site of hexokinase I. These mutants exhibited a 3-fold increase in Km for glucose but no change in either the Km for ATP or the kcat. The circular dichroism (CD) spectra showed no differences among the wild-type or mutant enzymes. These results suggest that Cys606 and Cys628 are not involved in glucose binding directly. The putative ATP-binding site of full-length human brain hexokinase may involve Arg539 and Gly679, and these residues were mutated to Ile. For the mutant R539I, the kcat value decreased 114-fold relative to wild-type hexokinase, whereas the Km values for ATP and glucose changed only slightly. No change was observed in the Ki value for 1,5-anhydroglucitol 6-phosphate. CD spectra showed only a slight change in secondary structure. For the mutant G679I, overexpressed hexokinase is insoluble. We suggest that Arg539 is important for catalysis because it stabilizes the transition state product ADP-hexokinase. Gly679 is probably important for proper folding of the protein.

Analysis of carbohydrate transport across the envelope of isolated cauliflower-bud amyloplasts.

Using isolated amyloplasts from cauliflower buds, we have characterized the interaction and transport of various carbohydrates across the envelope membrane of a heterotrophic plastid. According to our results, glucose 6-phosphate (Glc6P) and glucose 1-phosphate (Glc1P) do not share the same transport protein for uptake into cauliflower-bud amyloplasts. Glc6P-dependent starch synthesis is strongly inhibited in the presence of dihydroxyacetone phosphate (DHAP) or 4,4'-di-isothiocyano-2,2'- stilbenedisulphonic acid (DIDS), whereas Glc1P-dependent starch synthesis is hardly affected by these compounds. Analysis of the Glc6P uptake into proteoliposomes reconstituted from the envelope proteins of cauliflower-bud amyloplasts indicate that Glc6P is taken up in a counter-exchange mode with Pi, DHAP or Glc6P, whereas Glc1P does not act as a counter-exchange substrate. Pi is a strong competitive inhibitor of Glc6P uptake (Ki 0.8 mM) into proteoliposomes, whereas Glc1P does not significantly inhibit Glc6P transport. Beside a hexose-phosphate translocator, these amyloplasts possess an envelope protein mediating the transport of glucose across the membrane. This translocator exhibits an apparent Km for glucose of 2.2 mM and is inhibited by low concentrations of phloretin, known to be a specific inhibitor of glucose-transport proteins. Maltose inhibits the uptake of glucose (Ki 2.3 mM), indicating that both carbohydrates share the same translocator.

Human β-Cell Glucokinase: DUAL ROLE OF SER-151 IN CATALYSIS AND HEXOSE AFFINITY

Glucokinase is distinguished from yeast hexokinase and low Km mammalian hexokinases by its low affinity for glucose and its cooperative behavior, even though glucose binding residues and catalytic residues are highly conserved in all of these forms of hexokinase. The roles of Ser-151 and Asn-166 as determinants of hexose affinity and cooperative behavior of human glucokinase have been evaluated by site-directed mutagenesis, expression and purification of the wild-type and mutant enzymes, and steady-state kinetic analysis. Mutation of Asn-166 to arginine increased apparent affinity for both glucose and ATP by a factor of 3. Mutation of Ser-151 to cysteine, alanine, or glycine lowered the Km for glucose by factors of 2-, 26-, and 40-fold, respectively, decreased Vmax, abolished cooperativity for glucose, and also decreased Km for mannose and fructose. The Ser-151 mutants had hexose Km values similar to those of yeast hexokinase, hexokinase I, and the recombinantly expressed COOH-terminal half of hexokinase I. However, the Ki values for the competitive inhibitors, N-acetylglucosamine and glucose-6-P, were unchanged, suggesting that Ser-151 is not important for inhibitor binding. Mutation of Ser-151 also increased the Km for ATP about 5-fold and abolished the enzyme's low ATPase activity, which indicates it is essential for ATP hydrolysis. The substrate-induced change in intrinsic fluorescence of S151A occurred at a much lower glucose concentration than that for wild-type enzyme. The results implicate a dual role for Ser-151 as a determinant of hexose affinity and catalysis, exclusive of the glucose-induced conformational change, and suggest that the low hexose affinity of glucokinase is dependent on interaction of Ser-151 with other regions of the protein.

Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport

Zymomonas mobilis is known to transport glucose by a facilitated diffusion process. A putative glucose facilitator gene (glf), closely related to a large family of glucose transporters, is located in a cluster of genes that code for enzymes of glucose metabolism. The Z. mobilis glf gene is able to complement glucose transport in an Escherichia coli strain that is defective in native glucose transport and glucokinase. In this study, the recombinant E. coli was shown to be capable of influx counterflow when preloaded with glucose and had an apparent Km for glucose of approximately 1.1-2.9 mM, consistent with the function of Glf as a low-affinity glucose facilitator. The ability of glucokinase mutants expressing glf to transport glucose made it clear that glucokinase activity was not required for Glf-dependent glucose transport. The possibility that glucokinase can interact with Glf to improve the affinity for glucose was not supported since expression of the Z. mobilis glucokinase gene, in addition to glf, did not affect the Km of Glf for glucose in recombinant E. coli. The inability of various sugars to compete with glucose during glucose transport by recombinant E. coli expressing glf indicated that Glf is specific for glucose. While the results of fructose transport assays did not completely rule out the possibility of very low affinity for fructose, the apparent specificity of Glf for glucose makes it possible that Z. mobilis utilizes a different transporter(s) for fructose.

Site-directed mutagenesis studies on the determinants of sugar specificity and cooperative behavior of human beta-cell glucokinase.

The determinants of sugar specificity and cooperative behavior of human beta-cell glucokinase were studied by mutating several active site residues and performing a steady-state kinetic analysis of the purified mutant and wild-type enzymes after their expression in Escherichia coli. Asn-204, Glu-256, and Glu-290 were predicted from molecular modeling to interact with the 3-OH, 4-OH, 2-OH, and 1-OH groups of glucose. Mutation of these residues resulted in enzymes with decreased values of kcat and increased values of Km for glucose, mannose, and 2-deoxyglucose. Lys-56 is also predicted to make an interaction with the side chain of Glu-256 and its mutation increased the Km for glucose, deoxyglucose, mannose, and fructose by 4-, 4-, 3-, and 10-fold, respectively, and also increased the kcat for fructose by 5-fold. The Ki values for N-acetylglucosamine and mannoheptulose for the wild-type enzyme were 0.2 and 0.8 mM, respectively, and mutation of glucose binding residues to alanine resulted in an increase of about 3 orders of magnitude in these Ki values. Mutation of residues that directly hydrogen bond glucose hydroxyls (Asn-204, Glu-256, and Glu-290) to alanine resulted in enzymes that did not exhibit cooperative behavior, but mutation of Lys-56 or other residues that do not directly contact glucose had no effect on the Hill coefficient. Only glucose and deoxyglucose exhibited cooperative behavior. The results 1) confirm the predictions of the model that Asn-204, Glu-256, and Glu-290 are important residues involved in catalysis and hydrogen bonding glucose hydroxyl groups, 2) provide evidence for a role of Lys-56 in hexose binding, and 3) are consistent with the cooperative behavior of glucokinase being mediated by interactions of other regions of the protein with the highly conserved active site glucose binding residues.

Coexpression of glucose transporters and glucokinase in Xenopus oocytes indicates that both glucose transport and phosphorylation determine glucose utilization.

A Xenopus oocyte expression system was used to examine how glucose transporters (GLUT 2 and GLUT 3) and glucokinase (GK) activity affect glucose utilization. Uninjected oocytes and low rates of both glucose transport and phosphorylation; expression of GLUT 2 or GLUT 3 increased glucose phosphorylation approximately 20-fold by a low Km, endogenous hexokinase at glucose concentrations < or = 1 mM, but not at higher glucose concentrations. Coexpression of functional GK isoforms with GLUT 2 or 3 increased glucose utilization approximately an additional two- to threefold primarily at the physiologic glucose concentrations of 5-20 mM. The Km for glucose of both the hepatic and beta cell isoforms of GK, determined in situ, was approximately 5-10 mM when coexpressed with either GLUT 2 or GLUT 3. The increase in glucose utilization by coexpression of GLUT 3 and GK was dependent upon glucose phosphorylation since two missense GK mutations linked with maturity-onset diabetes, 182: Val-->Met and 228:Thr-->Met, did not increase glucose utilization despite accumulation of both a similar amount of immunoreactive GK protein and glucose inside the cell. Coexpression of a mutant GK and a normal GK isoform did not interfere with the function of the normal GK enzyme. Since the coexpression of GK and a glucose transporter in oocytes resembles conditions in the hepatocyte and pancreatic beta cell, these results indicate that increases in glucose utilization at glucose concentrations > 1 mM depend upon both a functional glucose transporter and GK.

Carbohydrate metabolism and physiology of the parasitic protist Trichomonas vaginalis studied in chemostats.

The parasitic protist Trichomonas vaginalis was cultured in chemostats with glucose or maltose as carbon and energy source. The maximum growth rate was about six divisions per day independent of the substrate, and the apparent Km for glucose was 0.375 mM. While growing on maltose, the growth rate depended linearly on the maltose concentration, indicating that in contrast to glucose metabolism a diffusion step is rate-limiting to maltose metabolism. Cultures were examined over a wide range of growth rates under four conditions: utilizing glucose or maltose as carbon and energy source, with the carbon source rate-limiting or present in excess. Cell density, cellular protein and carbohydrate content as well as residual substrate concentration in the culture fluid were measured at each steady state. The protein content was constant at 100 pg protein per cell except when T. vaginalis was cultured under glucose limitation; in the latter case, slow-growing cells had less protein than cells grown at high rates. When growing under glucose limitation T. vaginalis metabolism changed to become more energy efficient at growth rates exceeding about half the maximum rate. The maintenance energy at the low growth rates accounted for approximately half of the total carbon consumption, which is high in comparison to other micro-organisms. At low growth rates the yield on maltose exceeded that on glucose, when expressed in terms of carbon equivalents. The yields on maltose and glucose were equal, but much lower, when the carbon source was not rate-limiting.(ABSTRACT TRUNCATED AT 250 WORDS)

Sugar uptake by protoplasts of the ectomycorrhizal fungus, Amanita muse aria (L. ex fr.) Hooker

Protoplasts have been isolated from the ectomycorrhizal fungus, Amanita muscaria, for the first time. Yields were about 108 protoplasts per g fr. wt of mycelial suspension cultures treated with a combination of Novozym 234, chitinase and cellulase Onozuka R-10, and osmotically supplemented by 0-55 M sorbitol or 035 M KC1. Silicon oil filtration was used to investigate the uptake of metabolites by protoplasts using labelled compounds. About 66% of the initial protoplast population passed the oil layer after 50 s of centrifugation. Both glucose (as well as 3-O-methylglucose) and fructose were taken up by the protoplasts but not sucrose and mannitol. The Km for glucose (fructose) uptake was 1.25 mM (11.3), Vmax was 18 (30) pmoles (106 protoplasts min)-1 . Uptake of fructose was strongly inhibited by glucose but not vice versa; uptake of both was not inhibited by sucrose. The much higher affinity for glucose than for fructose suggests that glucose should be the main carbohydrate source for this fungus. Treatment of protoplasts with 10 μM antimycin A lowered cellular ATP/ADP ratios to well below 1 and significantly inhibited uptake of sugars.

Structure/function studies of human beta-cell glucokinase. Enzymatic properties of a sequence polymorphism, mutations associated with diabetes, and other site-directed mutants

Glucokinase plays a key role in the regulation of glucose metabolism in insulin-secreting pancreatic beta-cells and in the liver. Recent studies have shown that mutations in this enzyme can lead to the development of a form of non-insulin-dependent diabetes mellitus that is characterized by an autosomal dominant mode of inheritance and onset during childhood. Here, we report the catalytic properties of five additional missense mutations associated with diabetes (Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, Trp257-->Arg and Lys414-->Glu), one polymorphism present in both normal and diabetic subjects (Asp4-->Asn), and three site-directed mutations (Glu177-->Lys, Glu256-->Ala, and Lys414-->Ala). The Trp257-->Arg mutation generated an enzyme that had an activity that was less than 0.5% of that for native human beta-cell glucokinase. By contrast, the Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, and Lys414-->Glu mutations had a Vmax that was 20-100% of normal but a Km for glucose that was 8-14-fold greater than the native enzyme. There was no effect of the Asp4-->Asn polymorphism or the Glu177-->Lys substitution on glucokinase activity. The Lys414-->Ala substitution had no effect on Vmax but increased the Km for glucose 2-fold and the Glu256-->Ala substitution caused a approximately 200-fold decrease in Vmax. These studies have led to the identification of additional residues involved in glucokinase catalysis and substrate binding.

Glucose- and phorbol myristate acetate-stimulated oxygen consumption and superoxide production in rat peritoneal macrophages is inhibited by dexamethasone

1. Rat peritoneal macrophages stimulated with phorbol 12-myristate 13-acetate (PMA) (40 nM) show an increase in the rate of oxygen consumption (measured with an O2 electrode) and the production of superoxide (measured by cytochrome c reduction), which are both dependent on the presence of exogenous glucose. There is a 1:1 correlation between the oxygen consumed and the superoxide produced over a range of glucose concentrations (0-10 mM). 2. Preincubation of macrophages with dexamethasone (1 microM) for 3 h significantly decreased the Vmax. for PMA-induced glucose-dependent oxygen consumption (P < 0.001) and glucose-dependent superoxide production (P < 0.001). However, dexamethasone did not significantly change the Km for glucose in either PMA-induced oxygen consumption or superoxide production. Dexamethasone is therefore a non-competitive inhibitor of PMA-stimulated glucose-dependent oxygen consumption (Ki = 0.83 +/- 0.09 microM) and superoxide generation (Ki = 0.87 +/- 0.09 microM). 3. The PMA-induced rate of oxygen consumption by macrophages was decreased at oxygen concentrations below approx. 15 microM. The Km of oxygen for PMA-induced oxygen consumption was 1.28 +/- 0.13 microM (n = 12), and this was not significantly different in the presence of dexamethasone; Km = 1.61 +/- 0.31 microM (n = 12). It is therefore concluded that in vivo macrophage superoxide production is not limited by external oxygen or glucose concentrations, even in hypoxic joints.

Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships.

The glycolytic enzyme glucokinase plays an important role in the regulation of insulin secretion and recent studies have shown that mutations in the human glucokinase gene are a common cause of an autosomal dominant form of non-insulin-dependent (type 2) diabetes mellitus (NIDDM) that has an onset often during childhood. The majority of the mutations that have been identified are missense mutations that result in the synthesis of a glucokinase molecule with an altered amino acid sequence. To characterize the effect of these mutations on the catalytic properties of human beta-cell glucokinase, we have expressed native and mutant forms of this protein in Escherichia coli. All of the missense mutations show changes in enzyme activity including a decrease in Vmax and/or increase in Km for glucose. Using a model for the three-dimensional structure of human glucokinase based on the crystal structure of the related enzyme yeast hexokinase B, the mutations map primarily to two regions of the protein. One group of mutations is located in the active site cleft separating the two domains of the enzyme as well as in surface loops leading into this cleft. These mutations usually result in large reductions in enzyme activity. The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft. These mutations show a small approximately 2-fold reduction in Vmax and a 5- to 10-fold increase in Km for glucose. The characterization of mutations in glucokinase that are associated with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinase catalysis and localized functionally important regions of the glucokinase molecule.

Properties of the glucose phosphotransferase system of Clostridium acetobutylicum NCIB 8052

The glucose phosphotransferase system (PTS) of Clostridium acetobutylicum was studied by using cell extracts. The system exhibited a Km for glucose of 34 microM, and glucose phosphorylation was inhibited competitively by mannose and 2-deoxyglucose. The analogs 3-O-methylglucoside and methyl alpha-glucoside did not inhibit glucose phosphorylation significantly. Activity showed no dependence on Mg2+ ions or on pH in the range 6.0 to 8.0. The PTS comprised both soluble and membrane-bound proteins, which interacted functionally with the PTSs of Clostridium pasteurianum, Bacillus subtilis, and Escherichia coli. In addition to a membrane-bound enzyme IIGlc, sugar phosphorylation assays in heterologous systems incorporating extracts of pts mutants of other organisms provided evidence for enzyme I, HPr, and IIIGlc components. The HPr was found in the soluble fraction of C. acetobutylicum extracts, whereas enzyme I, and probably also IIIGlc, was present in both the soluble and membrane fractions, suggesting a membrane location in the intact cell.

Maltose transacetylase of Escherichia coli. Mapping and cloning of its structural, gene, mac, and characterization of the enzyme as a dimer of identical polypeptides with a molecular weight of 20,000

malQ mutants, lacking amylomaltase, cannot grown on maltose. However, when maltose is present in the medium, it can be accumulated to high internal levels. In a subsequent slow reaction, accumulated maltose becomes acetylated and leaks back into the medium. The enzyme responsible for this acetylation uses acetyl-CoA as acetyl donor and can be measured in crude extracts (Boos, W., Ferenci, T., and Shuman, H. A. (1981) J. Bacteriol. 146, 725-732). The structural gene for the enzyme, which we named mac, was mapped at 10.4 min on the Escherichia coli linkage map. We cloned a 3.4-kilobase pair PstI-EcoRI DNA fragment containing the mac gene. Cell-free extracts of a strain harboring the multicopy plasmid were used to purify the maltose-transacetylating activity to apparent homogeneity. On sodium dodecyl sulfate-polyacrylamide gels the enzyme exhibited a molecular weight of 20,000. Using molecular sieve chromatography, a molecular weight of 40,000 was determined for the native enzyme. Therefore, the enzyme is a dimer of two identical subunits. At a sugar concentration of 100 mM the enzyme acetylates glucose, maltose, mannose, galactose, and fructose in decreasing relative rate of 1, 0.55, 0.20, 0.07, 0.04. Maltotriose and other oligosaccharides were acetylated with 2% of the rate determined for glucose. The Km for glucose and maltose were 62 and 90 mM, and the Vmax was 0.20 and 0.11 mmol/min x mg enzyme, respectively.

Glucose phosphorylation. Site-directed mutations which impair the catalytic function of hexokinase.

Recent studies from this and other laboratories have resulted in the cloning and sequencing of hexokinases from a variety of tissues including yeast, human kidney, rat brain, rat liver, and mouse hepatoma. Significantly, studies on the hepatoma enzyme conducted in this laboratory (Arora, K.K., Fanciulli, M., and Pedersen, P.L. (1990) J. Biol. Chem. 265, 6481-6488) resulted also in its overexpression in Escherichia coli in active form. We have now used site-directed mutagenesis for the first time in studies of hexokinase to evaluate the role of amino acid residues predicted to interact with either glucose or ATP. Four amino acid residues (Ser-603, Asp-657, Glu-708, and Glu-742) believed to interact with glucose were mutated to alanine or glycine, whereas a lysine residue (Lys-558) thought to be directly involved in binding ATP was mutated to either methionine or arginine. Of all the mutations in residues believed to interact with glucose, the Asp-657----Ala mutation is the most profound, reducing the hexokinase activity to a level less than 1% of the wild type. The relative Vmax values for Ser-603----Ala, Glu-708----Ala, and Glu-742----Ala enzymes are 6, 10, and 6.5%, respectively, of the wild-type enzyme. Glu-708 and Glu-742 mutations increase the apparent Km for glucose 50- and 14-fold, respectively, while the Ser-603----Ala mutation decreases the apparent Km for glucose 5-fold. At the putative ATP binding site, the relative Vmax for Lys-558----Arg and Lys-558----Met enzymes are 70 and 29%, respectively, of the wild-type enzyme with no changes in the apparent Km for glucose. No changes were observed in the apparent Km for ATP with any mutation. These results support the view that all 4 residues predicted to interact with glucose from earlier x-ray studies may play a role in binding and/or catalysis. The Asp-657 and Ser-603 residues may be involved in both, while Glu-708 and Glu-742 clearly contribute to binding but are not essential for catalysis. In contrast, Lys-558 appears to be essential neither for binding nor catalysis.