Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho- forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis.

The mechanism by which cAMP-dependent protein kinase-catalyzed phosphorylation modulates the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was examined after site-specific mutation of the cAMP-dependent phosphorylation site (Ser32) to aspartic acid or alanine. The mutant and wild-type enzymes were overexpressed in Escherichia coli in a rich medium to levels as high as 30 mg/liter and were then purified to homogeneity. The kinetic properties of the Ser32-Ala mutant were identical with the dephosphorylated wild-type bifunctional enzyme. Mutation of Ser32 to aspartic acid mimicked several effects of cAMP-dependent phosphorylation: there was an increase in the Km for fructose 6-phosphate for 6-phosphofructo-2-kinase and an increase in the maximal velocity of fructose-2,6-bisphosphatase. Fructose-2,6-bisphosphatase activity of the Ser32-Asp mutant was 75% that of the phosphorylated wild-type enzyme, the mutant's kinase reaction had an identical dependence on fructose 6-phosphate, while its maximum velocity was only 60% that of the phosphorylated wild-type enzyme over a wide pH range. Furthermore, catalytic subunit-catalyzed in vitro phosphorylation of the Ser32-Ala mutant on Ser33 increased the Km for fructose 6-phosphate by 4-fold for the 6-phosphofructo-2-kinase. The results support the hypothesis that Ser32 is an important residue in the regulation of the activities of the bifunctional enzyme and that phosphorylation of Ser32 can be functionally substituted by aspartic acid. The results suggest a role for negative charge in the effect of phosphorylation.     

Amino acid sequence of the phosphorylation site of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae has properties similar to other gluconeogenic fructose-1,6-bisphosphatases, but an unusual characteristic of the yeast enzyme is that it can be phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation also occurs in vivo, presumably as part of a signalling mechanism for the enzyme's degradation. To probe the structural basis for the phosphorylation of yeast fructose-1,6-bisphosphatase, we have developed an improved procedure for the purification of the enzyme and then performed sequence studies with the in vitro-phosphorylated protein as well as with tryptic and chymotryptic peptides containing the phosphorylation site. As a result of these studies, we have determined that yeast fructose-1,6-bisphosphatase has the following 24-residue NH2-terminal amino acid sequence: Pro-Thr-Leu-Val-Asn-Gly-Pro-Arg-Arg-Asp-Ser-Thr-Glu-Gly- Phe-Asp-Thr-Asp-Ile-Ile-Thr-Leu-Pro-Arg. The site of phosphorylation is located at Ser-11 in the above sequence. The amino acid sequence around the site of phosphorylation contains the sequence - Arg-Arg-X-Ser- associated with many of the better substrates of cAMP-dependent protein kinase. The sequence of residues 15-24 above is highly homologous with the sequence of residues 6-15 of pig kidney fructose-1,6-bisphosphatase, showing 7 out of 10 residues in identical positions. The yeast enzyme, however, has a dissimilar NH2-terminal region which extends beyond the NH2 terminus of mammalian fructose-1,6-bisphosphatases and contains a unique phosphorylation site.     

Phosphorylation in vivo of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase at the cyclic AMP-dependent site

In vivo labeled fructose-1,6-bisphosphatase was immunopurified from yeast (Saccharomyces cerevisiae) cells that had been incubated in the presence of [32P] orthophosphate. Tryptic peptides from labeled enzyme were mapped by high performance liquid chromatography. Most of the radioactivity was found to be associated with the peptide Arg9 through Arg24, the same peptide which had been previously shown to be phosphorylated in vitro by cAMP-dependent protein kinase (Rittenhouse, J., Harrsch, P. B., Kim, J. N., and Marcus, F. (1986) J. Biol. Chem. 261, 3939-3943). The amino acid sequence analysis suggests that phosphorylation occurs at the same site, Ser11. We have also determined the extent of phosphorylation at Ser11 of fructose-1,6-bisphosphatase in yeast cultures growing under various nutritional conditions by measuring the relative amounts of phospho- and corresponding dephosphopeptides in tryptic digests. Significant levels of phosphorylation of the enzyme were found in yeast cultures grown under gluconeogenic conditions that varied from 0.15 to 0.50 mol of phosphate per mol of enzyme subunit. However, phosphate incorporation rapidly increased to greater than 0.8 mol after addition of glucose to these cultures. An alternative technique, based solely on enzyme activity measurements, was also developed to estimate the extent of fructose-1,6-bisphosphatase phosphorylation in yeast cultures. The results obtained with this technique agreed with those obtained by high performance liquid chromatography of tryptic peptides.     

Amino acid sequence similarity between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase

Chloroplast fructose-1,6-bisphosphatase is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars and the properties of this enzyme are clearly distinct from cytosolic gluconeogenic fructose-1,6-bisphosphatase. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. In the present study, purified spinach chloroplast fructose-1,6-bisphosphatase was reduced, S-carboxymethylated with iodoacetic acid, and cleaved with either cyanogen bromide or trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography. Automated Edman degradation of some of the purified peptides showed amino acid sequences highly homologous to residues 72-86, 180-199, and 277-319 of pig kidney fructose-1,6-bisphosphatase. These findings suggest a common evolutionary origin for mammalian gluconeogenic and chloroplast fructose-1,6-bisphosphatase, enzymes catalyzing the same reaction but having different functions and modes of regulation.     

Phosphorylation and inactivation of yeast fructose-1,6-bisphosphatase by cyclic AMP-dependent protein kinase from yeast

Purified fructose-1,6-bisphosphatase from Saccharomyces cerevisiae was phosphorylated in vitro by purified yeast cAMP-dependent protein kinase. Maximal phosphorylation was accompanied by an inactivation of the enzyme by about 60%. In vitro phosphorylation caused changes in the kinetic properties of fructose-1,6-bisphosphatase: 1) the ratio R(Mg2+/Mn2+) of the enzyme activities measured at 10 mM Mg2+ and 2 mM Mn2+, respectively, decreased from 2.6 to 1.2; 2) the ratio R(pH 7/9) of the activities measured at pH 7.0 and pH 9.0, respectively, decreased from 0.62 to 0.38, indicating a shift of the pH optimum to the alkaline range. However, the affinity of the enzyme for its inhibitors fructose-2,6-bisphosphate (Fru-2,6-P2) and AMP, expressed as the concentration required for 50% inhibition, was not changed. The maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase was 0.6-0.75 mol/mol of the 40-kDa subunit. Serine was identified as the phosphate-labeled amino acid. The initial rate of in vitro phosphorylation of fructose-1,6-bisphosphatase, obtained with a maximally cAMP-activated protein kinase, increased when Fru-2,6-P2 and AMP, both potent inhibitors of the enzyme, were added. As Fru-2,6-P2 and AMP did not affect the phosphorylation of histone by cAMP-dependent protein kinase, the inhibitors must bind to fructose-1,6-bisphosphatase in such a way that the enzyme becomes a better substrate for phosphorylation. Nevertheless, Fru-2,6-P2 and AMP did not increase the maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase beyond that observed in the presence of cAMP alone.     

Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase

Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.     

Amino acid sequence homology among fructose-1,6-bisphosphatases

The hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In the present studies, we have made a comparison of the primary structure of mammalian fructose-1,6-bisphosphatase with the sequence of peptides isolated from the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplast enzymes. Our results demonstrate a high degree of sequence homology, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.     

Changes of an androgen-dependent nuclear protein during functional differentiation and by dedifferentiation of the dorsolateral prostate of rats

In contrast with previous results that indicate that Saccharomyces cerevisiae fructose-1,6-bisphosphatase is a dimer of 56,000 molecular weight subunits, we find that the subunit Mr of the enzyme purified from baker's yeast is 40,000. The same subunit Mr was observed in immunoprecipitates of crude supernatants of baker's yeast and S. cerevisiae cultures, as well as in acid-extracts of cells detected by immunoblotting, suggesting that the native subunit indeed has a Mr of 40,000 and it has not been produced from a larger polypeptide. Complete immunoprecipitation of fructose-1,6-bisphosphatase activity with saturating concentrations of specific antibody suggests that there is only one fructose-1,6-bisphosphatase isozyme in S. cerevisiae. The Mr of the purified enzyme determined by size exclusion HPLC suggests that it has a tetrameric structure characteristic of fructose-1,6-bisphosphatases from a broad phylogenetic spectrum.     

Fructose 2,6-bisphosphate activates the cAMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase in vitro

Fructose-1,6-bisphosphatase purified from Saccharomyces cerevisiae is phosphorylated in vitro by a cAMP-dependent protein kinase. The phosphorylation reaction incorporates 1 mol of phosphate/mol of enzyme and is greatly stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate acts upon fructose-1,6-bisphosphatase, not on the protein kinase. The phosphorylation of fructose 1,6-bisphosphatase lowers its activity by about 50%. The characteristics of the phosphorylation reaction in vitro show that this modification is responsible for the inactivation of fructose-1,6-bisphosphatase observed in vivo.     

Regulation of fructose-1,6-bisphosphatase in yeast by phosphorylation/dephosphorylation

Fructose-1,6-bisphosphatase was precipitated with purified rabbit antiserum from extracts of ³²P-orthophosphate labelled yeast cells, submitted to SDS polyacrylamide gel electrophoresis, extracted from the gels and counted for radioactivity due to ³²P incorporation. Fructose-1,6-bisphosphatase from glucose starved yeast cells contained a very low ³²P label. During 3 min treatment of the glucose starved cells with glucose the ³²P-label increased drastically. Subsequent incubation of the cells in an acetate containing, glucose-free medium led to a label which was again low. Analysis for phosphorylated amino acids in the immunpprecipitated fructose-1,6-bisphosphatase protein from the 3 min glucose-inactivated cells exhibited phospho-serine as the only labelled phosphoamino acid. These data demonstrate a phosphorylation of a serine residue of fructose-1,6-bisphosphatase during this 3 min glucose treatment of glucose starved cells. A concomitant about 60 % inactivation of the enzyme had been shown to occur. The data in addition show a release of the esterified phosphate from the enzyme upon incubation of cells in a glucose-free medium, a treatment which leads to peactivation of enzyme activity. A protein kinase and a protein phosphatase catalysing this metabolic interconversion of fructose-1,6-bisphosphatase are postulated. It is assumed that metabolites accumulating after the addition of glucose exert a positive effect on the kinase activity and/or have a negative effect on the phosphatase activity. A role of the enzymic phosphorylation of fructose-1,6-bisphosphatase in the initiation of complete proteolysis of the enzyme during “catabolite inactivation” is discussed.     

Phosphorylation- and ligand-induced conformational changes of rat liver fructose-1,6-bisphosphatase

The effects of cyclic AMP-dependent phosphorylation on the structural properties of rat liver fructose-1,6-bisphosphatase were investigated by uv difference spectroscopy and circular dichroism. The incorporation of 4 mol of phosphate per mole of fructose-1,6-bisphosphatase induces a significant increase in the alpha-helix content of the enzyme without affecting its spectrophotometric properties. The addition of fructose 1,6-bisphosphate or fructose 2,6-bisphosphate also affects the conformation of the enzyme. However, both the phosphorylated and the nonphosphorylated forms exhibit similar ligand-induced conformational changes. These results show that cyclic AMP-dependent phosphorylation of fructose-1,6-bisphosphatase induces a specific conformational change. They also suggest that this modification does not alter the interaction of the enzyme protein with fructose 1,6-bisphosphate and fructose 2,6-bisphosphate.     

Ribose 1,5-bisphosphate inhibits fructose-1,6-bisphosphatase in rat kidney cortex

Fructose-1,6-bisphosphatase is one of the regulatory enzymes of gluconeogenesis in kidney cortex. The effect of ribose 1,5-bisphosphate on fructose-1,6-bisphosphatase purified from rat kidney cortex was studied. Rat kidney cortex, fructose-1,6-bisphosphatase exhibited hyperbolic kinetics with regard to its substrate, but the activity was inhibited by ribose 1,5-bisphosphate at nanomolar concentrations. The inhibitory effect of ribose 1,5-bisphosphate on the fructose-1,6-bisphosphatase was enhanced in the presence of AMP, one of the inhibitors of fructose-1,6-bisphosphatase. Fructose-2,6-bisphosphate, which is an inhibitor of fructose-1,6-bisphosphatase, inhibited rat kidney cortex fructose-1,6-bisphosphatase activities at a low concentration of fructose-1,6-bisphosphate but a high concentration of fructose-1,6-bisphosphate relieved fructose-1,6-bisphosphatase from fructose-2,6-bisphosphate-dependent inhibition. On the contrary, fructose-1,6-bisphosphate was not effective for the recovery of fructose-1,6-bisphosphatase from ribose 1,5-bisphosphate-dependent inhibition. These results suggest that ribose 1,5-bisphosphate is a potent inhibitor and is involved in the regulation of fructose-1,6-bisphosphatase in rat kidney cortex.     

The effect of high hydrostatic pressure on the modulation of regulatory enzymes from spinach chloroplasts.

High hydrostatic pressure enhanced the specific activity of regulatory enzymes of the Benson-Calvin cycle (fructose-1,6-bisphosphatase, glyceraldehyde-3-P dehydrogenase, phosphoribulokinase) which are modulated by the ferredoxin-thioredoxin system. High activity of chloroplast fructose-1,6-bisphosphatase required dithiothreitol, fructose 1,6-bisphosphate, and Ca2+. At 100 bar the A0.5 for fructose 1,6-bisphosphate (0.3 mM) was lower than that at 1 bar (1.5 mM), whereas similar variations of pressure did not alter the A0.5 for Ca2+ (55 microM). The response of chloroplast glyceraldehyde-3-P dehydrogenase exposed to 500 bar was a 4-fold increase in the NADP-linked activity; conversely, the NAD-dependent activity remained unchanged. The concerted action of high pressure and Pi (or ATP), both activators of chloroplast glyceraldehyde-3-P dehydrogenase, led to inactivation. On the other hand, the activity of phosphoribulokinase increased 10-fold when the enzyme was incubated at 1500 bar; the activation process was strictly dependent on the presence of dithiothreitol. At variance with these enzymes, bovine liver fructose-1,6-bisphosphatase, yeast glyceraldehyde-3-P dehydrogenase, and chloroplast ribulose 1,5-bisphosphate carboxylase, whose activities are not modulated by reduced thioredoxin, were inactivated by high pressure. The comparison of oligomeric enzymes revealed that the stimulation of specific activity by high pressure correlated with thioredoxin-mediated activation, and it did not depend on a particular subunit composition. Present results show that high pressure resembled thioredoxin, cosolvents, and chaotropic anions in its action on regulatory enzymes of the Benson-Calvin cycle. The comparison of physiological and non-physiological modulators suggested that thioredoxin-mediated modifications of noncovalent interactions is an important event in light-dependent regulation of chloroplast enzymes.     

Amino acid sequence of the phosphorylation site of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was phosphorylated by cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Treatment of the 32P-labeled enzyme with thermolysin removed all of the radioactivity from the enzyme core and produced a single labeled peptide. The phosphopeptide was purified by ion exchange chromatography, gel filtration, and reverse phase high pressure liquid chromatography. The sequence of the 12-amino acid peptide was found to be Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser(P)-Ser-Ile-Pro-Gln. Correlation of the extent of phosphorylation with activity showed that a 50% decrease in the ratio of kinase activity to bisphosphate activity occurred when only 0.25 mol of phosphate was incorporated per mol of enzyme subunit, and maximal changes occurred with 0.7 mol incorporated. The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of the native bifunctional enzyme was compared with that of other rat liver protein substrates. The Km for 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (10 microM) was less than that for rat liver pyruvate kinase (39 microM), fructose-1,6-bisphosphatase (222 microM), and 6- phosphofructose -1-kinase (230 microM). Comparison of the initial rate of phosphorylation of a number of protein substrates of the cyclic AMP-dependent protein kinase revealed that only skeletal muscle phosphorylase kinase was phosphorylated more rapidly than the bifunctional enzyme. Skeletal muscle glycogen synthase, heart regulatory subunit of cyclic AMP-dependent protein kinase, and liver pyruvate kinase were phosphorylated at rates nearly equal to that of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, while phosphorylation of fructose-1,6-bisphosphatase and 6-phosphofructo-1-kinase was barely detectable. Phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was not catalyzed by any other protein kinase tested. These results are consistent with a primary role of the cyclic AMP-dependent protein kinase in regulation of the enzyme in intact liver.     

Fructose-1,6-bisphosphatase from rat liver. A comparison of the kinetics of the unphosphorylated enzyme and the enzyme phosphorylated by cyclic AMP-dependent protein kinase

A purification procedure for rat hepatic fructose-1,6-bisphosphatase, described earlier, has been improved, resulting in an enzyme preparation with a neutral pH optimum and with both phosphorylatable serine residues present. The subunit Mr was 40,000. Phosphorylation in vitro with cyclic AMP-dependent protein kinase resulted in the incorporation of 1.4 mol of phosphate/mol of subunit and led to an almost 2-fold decrease in apparent Km for fructose-1,6-bisphosphate. In contrast to yeast fructose-1,6-bisphosphatase, fructose-2,6-bisphosphate had no effect on the rate of phosphorylation or dephosphorylation of the intact enzyme. The effects of the composition of the assay medium, with regard to buffering substance and Mg2+ concentration, on the apparent Km values of phosphorylated and unphosphorylated enzyme were investigated. The kinetics of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase were studied with special reference to the inhibitory effects of adenine nucleotides and fructose-2,6-bisphosphate. Unphosphorylated fructose-1,6-bisphosphatase was more susceptible to inhibition by both AMP and fructose 2,6-bisphosphate than phosphorylated enzyme, at high and low substrate concentrations. Both ATP and ADP had a similar effect on the two enzyme forms, ADP being the more potent inhibitor. Finally, the combined effect of several inhibitors at physiological concentrations was studied. Under conditions resembling the gluconeogenic state, phosphorylated fructose-1,6-bisphosphatase was found to have twice the activity of the unphosphorylated enzyme.     

Irreversible inactivation of Saccharomyces cerevisiae fructose-1,6-bisphosphatase independent of protein phosphorylation at Ser11

The fructose-1,6-bisphosphatase gene was used with multicopy plasmids to study rapid reversible and irreversible inactivation after addition of glucose to derepressed Saccharomyces cerevisiae cells. Both inactivation systems could inactivate the enzyme, even if 20-fold over-expressed. The putative serine residue, at which fructose-1,6-bisphosphatase is phosphorylated, was changed to an alanine residue without notably affecting the catalytic activity. No rapid reversible inactivation was observed with the mutated enzyme. Nonetheless, the modified enzyme was still irreversibly inactivated, clearly demonstrating that phosphorylation is an independent regulatory circuit that reduces fructose-1,6-bisphosphatase activity within seconds. Furthermore, irreversible glucose inactivation was not triggered by phosphorylation of the enzyme.     

Regulation of fructose-1,6-bisphosphatase activity in primary cultured astrocytes

In the gluconeogenic pathway, fructose-1,6-bisphosphatase (EC 3.1.3.11) is the last key-enzyme before the synthesis of glucose-6-phosphate. The extreme diversity of cells present in the whole brain does not facilitate in vivo study of this enzyme and makes it difficult to understand the regulatory mechanisms of the related carbohydrate metabolism. It is for instance difficult to grasp the actual effect of ions like potassium, magnesium and manganese on the metabolic process just as it is difficult to grasp the effect of different pH values and the influence of glycogenic compounds such as methionine sulfoximine. The present investigation attempts to study the expression and regulation of fructose-1,6-bisphosphatase in cultured astrocytes. Cerebral cortex of new-born rats was dissociated into single cells that were then plated. The cultured cells were flat and roughly polygonal and were positively immunostained by anti-glial fibrillary acidic protein antibodies. Cultured astrocytes are able to display the activity of fructose-1,6-bisphosphatase. This activity was much higher than that in brain tissue in vivo. Fructose-1,6-bisphosphatase in cultured astrocytes did not require magnesium ions for its activity. The initial velocity observed when the activity was measured in standard conditions was largely increased when the enzyme was incubated with Mn²⁺. This increase was however followed by a decrease in absorbance resulting in the induction, by the manganese ions, of a singular kinetics in the enzyme activity. Potassium ions also stimulated fructose-1,6-bisphosphatase activity. When the enzyme was exposed to different pH values ranging from 6 to 9 units, the highest activity was observed at pH 6. When the cultured astrocytes were incubated with methionine sulfoximine, the fructose-1,6-bisphosphatase activity increased. This increase was quick and depended on the dose of methionine sulfoximine. These results show that cultured astrocytes are able to maintain fructose-1,6-bisphosphatase activity. With the exception of the higher level activity associated acidic pH ranges, the properties of the enzyme resemble those of the in vivo enzyme. Methionine sulfoximine has a direct effect on astrocytes in its activation of fructose-1,6-bisphosphatase. It is concluded that the expression and the regulation of fructose-1,6-bisphosphatase activity in cultured astrocytes look like those in the brain. Astrocytes are probably the principal cells that express this activity in the brain in vivo.     

Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes.

A new over-expression system has been set up for Escherichia coli thioredoxin, yielding 55 mg purified protein/10 g fresh cells. This system has been used to produce thioredoxin modified by site-directed mutagenesis. Taking advantage of the structural and enzymatic similarity between E. coli and spinach m-type thioredoxin, Asp61 of E. coli thioredoxin has been changed into Asn in order to investigate the impact of the suppression of a charged residue on the interaction of thioredoxin with target enzymes. The modification did not significantly alter the structure of the protein. Neither the rate of reduction of insulin and 5,5'-dithio-bis(2-nitrobenzoic acid) by the reduced thioredoxin, nor the reduction by NADPH-dependent thioredoxin reductase, have been modified. The major effect of the mutation was observed for chloroplast enzyme activation with thioredoxin reduced by dithiothreitol and with thioredoxin reduced by ferredoxin-dependent thioredoxin reductase in a light-activation reconstituted chloroplast system. The substitution of the negatively charged Asp61 by the neutral Asn led to an increase in the efficiency of spinach fructose-1,6-bisphosphatase activation by the dithiothreitol-reduced thioredoxin, and to an increase in both spinach fructose-1,6-bisphosphatase and corn NADP-dependent malate dehydrogenase activities in the light-activation system. This suggests that the suppression of the negative charge improves the reactivity of thioredoxin with chloroplast enzymes such as fructose-1,6-bisphosphatase and ferredoxin-dependent thioredoxin reductase.     

Functioning of fructose-1,6-bisphosphatase--main enzyme of gluconeogenesis in microorganisms

The modern literature data about common characteristics, genetic and molecular-biological properties of main enzyme of gluconeogenesis (fructose-1,6-bisphosphatase) were analyzed. Regulation of fructose-1,6-bisphosphatase activity (stimulation and inhibition) by fructose-1,6-bisphosphate, fructose-2,6-bisphosphate, phosphoenolpyruvate, AMP and by metal ions are discussed. It was concluded that apart from the fact that fructose-1,6-bisphosphatase was intensively investigated, this enzyme from Mollicutes failed to be studied sufficiently.