Temperature dependency of the anomeric specificity of yeast and bovine hexokinases

The phosphorylation of alpha- and beta-D-glucose by either yeast hexokinase or beef heart hexokinase was measured at both 10 and 30 degrees C. At 30 degrees C, the anomeric specificity of yeast hexokinase represented a mirror image of that of bovine hexokinase, in terms of both maximal velocity and affinity. A decrease in temperature apparently accentuated the anomeric difference in both maximal velocity and affinity of bovine hexokinase. Such a difference consisted in a higher maximal velocity with beta- than alpha-D-glucose, but a greater affinity for the alpha- than beta-anomer. In yeast hexokinase, however, the decrease in temperature suppressed the anomeric difference in maximal velocity and inversed the anomeric difference in affinity. In the case of both enzymes, the fall in temperature decreased more the maximal velocity recorded with alpha-D-glucose than that measured with beta-D-glucose, and severely lowered the Km for alpha-D-glucose, whilst failing to affect significantly the Km for beta-D-glucose. These findings, which allow to reconcile prior apparently conflicting data, reveal that the anomeric behaviour of hexokinase is affected by the ambient temperature. Our data also support the view that hexokinase underwent a phylogenic evolution in terms of its anomeric specificity.     

Phosphorylation of yeast hexokinases

We show by the use of 32P-labeling in vivo that hexokinase 2 and hexokinase 1 in Saccharomyces cerevisiae are phosphoproteins. The highest labeling was after incubation in medium with a low concentration of glucose, when labeling appears to be predominant even without use of immunoprecipitation. The nature of the modification is not known, but it has properties consistent with a phosphomonoester of serine or threonine. The cAMP-dependent protein kinase plays a negative role in hexokinase phosphorylation, in that there was reduced labeling in strains (bcy1) lacking a regulatory subunit, and increased labeling during growth with high concentrations of glucose in a strain attenuated in the catalytic subunit (tpk1w1). The function of the modification is not known, but there was a correlation between the extent of labeling and the expression of kinase-dependent high-affinity glucose uptake.     

Phosphorylation by liver glucokinase of D-glucose anomers at anomeric equilibrium

The relative contribution of each anomer of D-glucose to the overall phosphorylation rate of the hexose tested at anomeric equilibrium was examined in rat liver postmicrosomal supernatants under conditions aimed at characterizing the activity of glucokinase, with negligible interference of either hexokinase, N-acetyl-D-glucosamine kinase or glucose-6-phosphatase (acting as a phosphotransferase). Both at 10 degrees and 30 degrees C, the relative contribution of each anomer was unaffected by the concentration of D-glucose. At both temperatures, the alpha/beta ratio for the contribution of each anomer was slightly, but significantly, lower than the alpha/beta ratio of anomer concentrations. These findings, which are consistent with the anomeric specificity of glucokinase in terms of affinity, cooperativity and maximal velocity, reveal that the preferred alpha-anomeric substrate for both glycogen synthesis and glycolysis is generated by glucokinase at a lower rate than is beta-D-glucose-6-phosphate.     

Purine nucleoside diphosphate regulation of yeast hexokinases.

The low activity state of hexokinase P-II, originally produced by Kosow and Rose by lowering the pH from 8 to 7 in certain sulfonated buffers, is not observed in Tris or imidazole buffers at pH 7 unless low concentrations of ADP or GDP are added. At pH values below 7 in imidazole buffer, partial inhibition occurs by protonation alone, and ADP or GDP causes further inhibition. As in the Kosow-Rose experiments, the enzyme in the low activity state can be activated either by excess ATP or by low concentrations of citrate, 3-phosphoglycerate and other metabolites. The inhibition by nucleoside diphosphates is greater at high glucose concentration. Hexokinase P-I is much less susceptible to regulation by nucleoside diphosphates or citrate, suggesting different physiological roles for the two isoenzymes.     

Synthesis of sterically crowded derivatives of anomeric pairs of D-glucose disaccharides

Derivatization of carbohydrates is of considerable interest since the derivatives can be used for structural studies in the field of mass spectrometry. We report here the synthesis of a series of sterically crowded derivatives of various linkage and stereo-isomeric glucose-glucose disaccharides with the impetus being to understand the effect of these derivatized groups on fragmentation of the glycosidic bond and the development of methodology for discernment of the anomeric configuration. The synthesis of per-alkylated (methyl, ethyl, propyl, butyl, and pentyl), per-esterified (acetyl, pivaloyl, mesitoyl), and per-silylated (tert-butyl-dimethyl silyl) glucose--glucose disaccharide derivatives has been reported.     

Purification and serological comparison of the yeast hexokinases P-I and P-II

An improved procedure for purification of the hexokinases P-I and P-II from baker's yeast is described. Yields, reproducibility, and purity are improved over those found by the methods used previously in this laboratory. The growth of large crystals of form P-I is described.Antisera prepared against the two purified hexokinases show only slight cross reaction by microcomplement fixation. The anti-sera have been used to demonstrate the presence of both P-I and P-II in crude extracts of various yeasts, including two haploid strains, and their absence in a yeast which contains glucokinase but no hexokinases.     

Catalytic activity with associated and dissociated forms of the yeast hexokinases.

In agreement with previous glucose binding data, kinetic studies of the yeast hexokinases at high protein concentration show that the dimeric forms P-I and P-II require much higher glucose concentrations for half-maximal rates than do the monomeric forms S-I and S-II, but each P form reaches the same V value as the corresponding S form. Raising the temperature from 5 °C to 24 °C causes an apparent dissociation of the P forms, most pronounced in the case of P-I, as evidenced by a lowering of glucose K_M values toward those of the S forms. Similarly, kinetic measurements at low protein concentrations, where the P forms are presumably fully dissociated, give identical glucose K_M values for the P and S forms.     

Relaxation spectra of yeast hexokinases. Isomerization of the enzyme.

Yeast hexokinase isozymes P1 and P11 exhibit a pH dependent, rapid relaxation process at 15 degrees C at enzyme concentrations of 100-474 muM and over a pH range of 6-8. The process was detected by equilibrium temperature jump spectroscopy using the indicator probe phenol red. The value of 1/tau varies from about 6 ms-1 at pH 8 for both isozymes to 50 ms-1 for P1 and 85 ms-1 for P11 at pH 6. The data are consistent with a mechanism involving an enzyme isomerization coupled to an ionization. The forward rate constant for the isomerization of the proposed mechanism varies between 3 and 7 ms-1; the ratio of the reverse rate constant to the ionization Ka is between 0.5 and 2 X 10(11) M-1 S-1; the estimated pKa varies between 5.5 and 6.1. The ranges of values in rate constants and pKa represent variations observed between preparations of the same isozyme and between isozymes. The isomerization rate is at least 50 times faster than catalysis under all conditions and the pKa is lower than that controlling activity. The rate of isomerization is unchanged by addition of sugar and nucleotide ligands, but the amplitude of the process is perturbed. These data imply that isomerizing and ionizing forms are sensitive to events at the active site. These equilibria between forms of hexokinase are fast enough, and have the right properties, to be important to the mechanism and regulation of the enzyme.     

Identification of a peptide sequence involved in association of subunits of yeast hexokinases

The chemistry of the proteolytic conversion of the native yeast hexokinases P-I and P-II to the respective modified forms S-I and S-II was studied in detail. The conversion of P-I to S-I was found to involve the removal of one six and one five residue peptide from P-I; these peptides were isolated and sequenced, and a comparison with the partial sequence of native P-I demonstrated that they were cleaved from the amino terminal end. Since results indicated that exactly the same peptides were cleaved from P-II during conversion to S-II, it is concluded that the first 11 amino acids in P-I and P-II have the same sequence. That sequence is: val · his · leu · gly · pro · lys · lys · pro · gln · ala · arg The basicity of these peptides was reflected in the decrease in isolectric point observed when a P-form is converted to an S-form. The peptides are clearly involved in the association of the subunits of yeast hexokinase, since their removal greatly weakens the tendency of the subunits to dimerize.     

Kinetics of glucose isomerization to fructose by immobilized glucose isomerase: anomeric reactivity of D-glucose in kinetic model

The substrate specificity of immobilized D-glucose isomerase (EC 5.3. 1.5) is investigated with an immobilized enzyme-packed reactor. A series of isomerization experiments with alpha-, beta-, and equilibrated D-glucose solutions indicates that beta anomer as well as alpha anomer is a substrate of the glucose isomerase at pH 7.5 and 60 degrees C. For substrate concentration of 0.028 mol l(-1) (1% w/v), the initial conversion rate of alpha-D-glucose was 43% higher than that with equilibrated glucose at the same concentration and 113% higher than beta-D-glucose conversion rate. This anomeric reactivity of glucose isomerase is mathematically described with a set of kinetic equations based on the reaction steps complying with Briggs-Haldane mechanism and the experimentally determined kinetic constants. The proposed reaction mechanism includes the mutarotation and the isomerization reactions of alpha- and beta-D-glucose with different rate constants.     

Enzyme activities of D-glucose metabolism in the fission yeast Schizosaccharomyces pombe.

The activities of key enzymes that are members of D-glucose metabolic pathways in Schizosaccharomyces pombe undergoing respirative, respirofermentative, and fermentative metabolisms are monitored. The steady-state activities of glycolytic enzymes, except phosphofructokinase, decrease with a reduced efficiency in D-glucose utilization by yeast continuous culture. On the other hand, the enzymic activities of pentose monophosphate pathway reach the maximum when the cell mass production of the cultures is optimum. Enzymes of tricarboxylate cycle exhibit the maximum activities at approximately the washout rate. The steady-state activity of pyruvate dehydrogenase complex increases rapidly when D-glucose is efficiently utilized. By comparison, the activity of pyruvate decarboxylase begins to increase only when ethanol production occurs. Depletion of dissolved oxygen suppresses the activity of pyruvate dehydrogenase complex but facilitates that of pyruvate decarboxylase. Acetate greatly enhances the acetyl CoA synthetase activity. Similarly, ethanol stimulates alcohol dehydrogenase and aldehyde dehydrogenase activities. Evidence for the existence of alcohol dehydrogenase isozymes in the fission yeast is presented.     

Glucose 6-phosphate release of wild-type and mutant human brain hexokinases from mitochondria

One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require approximately 7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.     

Biosynthesis of D-mannitol from D-glucose by Aspergillus candidus

Mannitol has long been known as a product of glucose metabolism by some strains of Aspergillus. Apparently no concerted effort, has been made to develop a practical fermentation process to make mannitol. Work at the Northern Laboratory has shown that nearly all strains of white Aspergillus produce significant amounts of mannitol; many strains of black Aspergillus also have this characteristic. Aspergillus candidus NRRL 305 is an exceptionally good mannitol producer. Studies on a fermentation process were conducted in 20-1, stainless steel fermentors, without baffles. Czapek-Dox medium, modified by addition of corn meal, yeast extract, and enzymatically hydrolyzed casein was the most satisfactory medium tested. Suitable increments of glucose were fed daily to the fermentors. The duration of the fermentation was from 10 to 16 days. The effects of agitation, aeration, temperature, and pH of the medium were studied. Under optimal conditions yields of mannitol approached 50% of the glucose consumed.     

The anomeric form of D-fructose 1,6-bisphosphate used as substrate in the muscle and yeast aldolase reactions.

From a series of rapid quench kinetic experiments, it has been demonstrated that muscle D-fructose bisphosphate aldolase catalyzes the cleavage of beta-D-fructose 1,6-bisphosphate but not that of the alpha anomer, although the alpha anomer may be tightly bound. Yeast D-fructose bisphosphate aldolase appears to utilize both alpha and beta anomers of the substrate, with yeast apoaldolase catalyzing the interconversion of the alpha and beta forms.