Catabolite inactivation of isocitrate lyase from Saccharomyces cerevisiae

A reversible carbon catabolite inactivation step is described for isocitrate lyase from Saccharomyces cerevisiae. This reversible inactivation step of isocitrate lyase is similar to that described for fructose 1,6-bisphosphatase. Addition of 2,4-dinitrophenol, nystatin or glucose to cultures, grown in ethanol as carbon source, caused a rapid loss of the isocitrate lyase and fructose 1,6-bisphosphatase activities at pH 5.5 but not at pH 7.5. These results suggest that intracellular acidification and thus a cAMP increase is involved in the catabolite inactivation mechanism of both enzymes. From results obtained by addition of glucose to yeast cultures at pH 7.5 it was concluded that others factors than cAMP can play a role in the catabolite inactivation mechanism of both enzymes.     

Periodate-oxidized AMP as a substrate, an inhibitor and an affinity label of human placental alkaline phosphatase.

1. Addition of glucose induced an inactivation of mitochondrial enzymes in the yeast Saccharomyces cerevisiae containing normal mitochondrial particles. 2. The glucose-induced inactivation of mitochondrial enzymes was inhibited by the presence of cycloheximide. 3. Pepstatin also inhibited the inactivation, but phenylmethanesulphonyl fluoride accelerated the inactivation. 4. The specific activities of fructose 1,6-bisphosphatase and cytoplasmic malate dehydrogenase were decreased on the exposure to glucose, as well as those of the mitochondrial enzymes. However, the glucose-induced inactivation of cytoplasmic enzymes was not inhibited by the presence of pepstatin. 5. The specific activities of hexokinase and phosphofructokinase, which are cytoplasmic enzymes were increased by the addition of glucose, and this effect was not affected by pepstatin. 6. Addition of glucose resulted in an increase in the synthesis of proteins of the mitochondria and the cytosol, and simultaneously in degradation of these mitochondrial and cytoplasmic proteins.     

Catabolite inactivation of fructose 1,6-bisphosphatase inKluyveromyces fragilis

Catabolite inactivation of fructose 1,6-bisphosphatase inKluyveromyces fragilis was found to occur as a one-step process with a half-life of approximately 90 min in contrast to the two-step process previously reported forSaccharomyces cerevisiae. No rapid initial 50% loss of activity immediately after a glucose-induced catabolite inactivation was found; nevertheless, fructose 1,6-bisphosphatase was rapidly phosphorylated within 5 min of glucose addition. This result supports the hypothesis that protein phosphorylation serves as a signal for the specific degradation of fructose 1,6-bisphosphatase during catabolite inactivation.     

The use of phenylmethylsulfonyl fluoride in the study of catabolite inactivation and repression in intact cells of Saccharomyces cerevisiae

Catabolite inactivation of fructose-1,6-bisphosphatase, isocitrate lyase, phosphoenolpruvate carboxykinase and malate dehydrogenase in intact cells could be prevented by phenylmethylsulfonyl fluoride added 40 min prior to the addition of glucose. Protein synthesis, fermentative and respiratory activity and catabolite repression were not affected. Elimination of catabolite inactivation by the addition of PMSF revealed that catabolite repression started at different times for different enzyme.Abbreviation PMSF phenylmethylsulfonyl fluoride     

Inactivation of gluconeogenic enzymes in glycolytic mutants of Saccharomyces cerevisiae.

Yeast mutants blocked at different steps of the glycolytic pathways have been used to study the inactivation of several gluconeogenic enzymes upon addition of sugars. While phosphorylation of the sugars appears a requisite for the inactivation of fructose 1,6-bisphosphatase and phosphoenol-pyruvate carboxykinase, malate dehydrogenase is inactivated by fructose in mutants lacking hexokinase. The normal inactivation elicited by glucose in a mutant lacking phosphofructokinase indicates that the process does not require metabolism of the sugar beyond hexose monophosphates. A possible role for ATP in the inactivation process is suggested.     

Cyclic AMP,fructose-2,6-bisphosphate and catabolite inactivation of enzymes in the hydrocarbon-assimilating yeast Candida maltosa

The inactivation of fructose-1,6-bisphosphatase, isocitrate lyase and cytoplasmic malate dehydrogenase in Candida maltosa was found to occur after the addition of glucose to starved cells. The concentration of cyclic AMP and fructose-2,6-bisphosphate increased drastically within 30 s when glucose was added to the intact cells of this yeast. From these results it was concluded that catabolite inactivation, with participation of cyclic AMP and fructose-2,6-bisphosphate, is an important control mechanism of the gluconeogenetic sequence in the n-alkane-assimilating yeast Candida maltosa, as described for Saccharomyces cerevisiae.     

Evidence for non-vacuolar proteolytic catabolite inactivation of yeast fructose-1,6-bisphosphatase

Immunoblotting was used to study whether proteolytic degradation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in yeast cells during catabolite inactivation occurs intra- or extravacuolarly. The 40-kDa subunits of both the phosphorylated and the non-phosphorylated fructose-1,6-bisphosphatase are rapidly degraded by an extract from isolated vacuoles to a 32-kDa intermediate which accumulates and is then slowly further degraded. However, in intact cells, neither the 32-kDa nor any other intermediate reacting with the fructose-1,6-bisphosphatase antibodies is observed following glucose-induced degradation of the enzyme. These observations are discussed as evidence against intravacuolar degradation of fructose-1,6-bisphosphatase during proteolytic catabolite inactivation.     

Studies on rapid reversible and non-reversible inactivation of fructose-1,6-bisphosphatase and malate dehydrogenase in wild-type and glycolytic block mutants of Saccharomyces cerevisiae

Experimental conditions have been elaborated to test for reversibility of the malate dehydrogenase inactivation (E.C.1.1.1.37) after addition of glucose to derepressed yeast cells. Malate dehydrogenase inactivation was shown to be irreversible at all stages of inactivation. In contrast fructose-1,6-bisphosphatase inactivation (E.C.3.1.11) remained reversible for at least 30 min after addition of glucose. Rapid reversible inactivation of fructose-1,6-bisphosphatase and irreversible inactivation of malate dehydrogenase were additionally investigated in glycolytic block mutants. Normal inactivation kinetics were observed in mutants without catalytic activity of phosphoglucose isomerase (E.C.5.3.1.9), phosphofructokinase (E.C.2.7.1.11), triosephosphate isomerase (E.C.5.3.1.1) and phosphoglycerate kinase (E.C.2.7.2.3). Hence, neither type of inactivation depended on the accumulation of any glucose metabolite beyond glucose-6-phosphate. Under anaerobic conditions irreversible inactivation was completely abolished in glycolytic block mutants. In contrast rapid reversible inactivation was independent of energy provided by respiration or fermentation. Reversibility of fructose-1,6-bisphosphatase inactivation was tested under conditions which prevented irreversible malate dehydrogenase inactivation. In these experiments, fructose-1,6-bisphosphatase inactivation remained reversible for at least 120 min, whereas reversibility was normally restricted to about 30 min. This indicated a common mechanism between the irreversible part of fructose-1,6-bisphosphatase inactivation and irreversible malate dehydrogenase inactivation.     

Studies on glucose-induced inactivation of gluconeogenetic enzymes in adenylate cyclase and cAMP-dependent protein kinase yeast mutants

Glucose-induced inactivation of the gluconeogenetic enzymes fructose-1,6-biphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase was tested in yeast mutants defective in adenylate cyclase (cyr1 mutation) and in the cAMP-binding subunit of cAMP-dependent protein kinase (bcy 1 mutation). In the mutant AM7-11D (cyr1 mutation), glucose-induced cAMP overshoot was absent, and no significant inactivation of the gluconeogenetic enzymes was detected, thus supporting the role of cAMP in the process. Moreover, in the mutant AM9-8B (bcy1 mutation), no cAMP-dependent protein kinase activity was evidenced, and, in addition, a normal inactivation pattern was observed, thus indicating that other mechanisms evoked by glucose might be required in the process. In the double mutant AM7-11DR-4 (cyr1 bcy1 mutations), no inactivating effect was triggered by the sugar: this suggests that cAMP exerts some additional effect on the process, besides the activation of cAMP-dependent protein kinase. Furthermore, in AM7-11D, extracellular cAMP triggered about 50% of inactivation of fructose-1,6-bisphosphatase; this effect was largely reversed in acetate medium plus cycloheximide even after 150 min of incubation. However, an extensive and essentially irreversible inactivation was evidenced in the presence of glucose plus cAMP, whereas glucose alone was only slightly effective. Therefore, the reversible effect of cAMP, which probably corresponds to enzyme phosphorylation, seems to be required for the irreversible, probably proteolytic, glucose-stimulated inactivation of this enzyme. Cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase in AM7-11D were also inactivated by cAMP, and much more by glucose plus cAMP, whereas glucose was practically ineffective. However, reversibility of the effect was not detected, and, in addition, no phosphorylation of phosphoenolpyruvate carboxykinase could be evidenced. Therefore, the sugar quite probably stimulates proteolysis of these enzymes, but the mechanism of cAMP in their degradation has still to be defined.     

Catabolite inactivation,cyclic AMP and protein phosphorylation in the methylotrophic yeastHansenula polymorpha

The inactivation of the peroxisomal enzyme alcohol oxidase and the cytoplasmic enzymes fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase was found to occur after addition of glucose to methanol-grown cells of the yeastHansenula polymorpha. The concentration of cyclic AMP increased nearly twofold within 3 min under the same conditions. In crude extracts ofH. polymorpha about 20 proteins are phosphorylated by cyclic AMP dependent protein kinases, among them also fructose-1,6-bisphosphatase. No phosphorylation of the alcohol oxidase protein could be detected. From this fact, it was concluded that the inactivation of the peroxisomal alcohol oxidase is independent of cyclic AMP-dependent protein phosphorylation.     

Characterization of a mutant of the yeast Candida maltosa defective in catabolite inactivation of gluconeogenetic enzymes

A spontaneous mutant of the yeast Candida maltosa SBUG 700 was isolated showing pseudohyphal marphology under all growth conditions tested. The C. maltosa PHM mutant takes up glucose with the kinetics of C. maltosa SBUG 700 and starved cells contain the same cyclic AMP concentration. Addition of glucose to the PHM mutant does not result in an increase of the intracellular cyclic AMP level and in catabolite inactivation of fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. However, addition of 2,4-dinitrophenol is followed by a rapid, transient increase of the cyclic AMP level in the mutant cells, but not by catabolite inactivation. These results show that a common mechanism might be responsible for catabolite inactivation and glucose-induced cAMP signaling or that glucose-induced cAMP signaling is required for catabolite inactivation in C. maltosa.     

Differential sensitivities to glucose and galactose repression of gluconeogenic and respiratory enzymes from Saccharomyces cerevisiae

The synthesis of isocitrate lyase was induced by the presence of ethanol in the chemostat reaching a specific activity of 200 mU·mg^-1 at this induced state. In glucoselimited, derepressed cells, 20 mU·mg^-1 were detected and under repressed conditions isocitrate lyase activity was not detected.The sensitivity of gluconeogenic enzymes: cytoplasmic malate dehydrogenase; fructose 1,6-bisphosphatase and isocitrate lyase as well as the mitochondrial enzymes NADH dehydrogenase and succinate cytochrome c oxidase to glucose and galactose repression were studied in chemostat cultures. Our results show that galactose was less effective as a repressor than glucose. Malate dehydrogenase was completely inactivated by glucose, whereas galactose only produced a 78% decrease of specific activity. Fructose 1,6-bisphosphatase and isocitrate lyase were completely inactivated by both sugars but at different rate. Glucose produced an 85% decrease of specific activity of the mitochondrial enzymes whereas galactose only decrease an 67%.     

Initiation of selective proteolysis by metabolic interconversion

After the addition of glucose to acetate- or ethanol-grown yeast cells a small group of selected enzymes is rapidly inactivated. This phenomenon has been called "catabolite inactivation". Among other enzymes participating in gluconeogenesis, fructose-1,6-bisphosphatase is inactivated during this catabolite inactivation process. It was shown by FUNAYAMA et al. (Eur. J. Biochem. 109, 61-66 (1980)) that the mechanism of inactivation is proteolysis. In the present paper evidence is presented that after addition of glucose a covalent conversion of the enzyme protein by phosphorylation of a serine-residue initiates its subsequent proteolysis. It is suggested that the covalent modification triggered by glucose and/or products of its catabolism renders the enzyme susceptible to proteinases and thereby initiates proteolysis of a selected enzyme without the necessity of a specific proteinase present.     

Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate.

Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.     

Metabolic effects of benzoate and sorbate in the yeast Saccharomyces cerevisiae at neutral pH

Preincubation of yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6.8 elicited a set of metabolic effects on sugar metabolism, which became apparent after the subsequent glucose addition. They can be summarized as follows: a) reduced glucose consumption; b) inhibition of glucose- and fructose-phosphorylating activities; c) supression of glucose-triggered peak of hexoses monophosphates; d) substantial reduction of glucose-triggered peak of fructose 2,6-bisphosphate; e) block of catabolite inactivation of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, but not of cytoplamic malate dehydrogenase. On the whole this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenetic to a glycolytic state. Our data also show that, unlike former assumptions, intracellular acidification is not likely to mediate the bulk of metabolic effects of benzoate and sorbate, since under our working conditions intracellular pH kept close to neutrality.     

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.     

Degradation of endogenous fructose during catabolism of sucrose and mannitol in halophilic archaebacteria

Metabolism of fructose arising endogenously from sucrose or mannitol was studied in halophilic archaebacteria Haloarcula vallismortis and Haloferax mediterranei. Activities of the enzymes of Embden-Meyerhof-Parnas (EMP) pathway, Entner-Doudoroff (ED) pathway and Pentose Phosphate (PP) pathway were examined in extracts of cells grown on sucrose or mannitol and compared to those grown on fructose and glucose. Sucrase and NAD-specific mannitol dehydrogenase were induced only when sucrose or mannitol respectively were the growth substrates. Endogenously arising fructose was metabolised in a manner similar to that for exogenously supplied fructose i.e. a modified EMP pathway initiated by ketohexokinase. While the enzymes for modified EMP pathway viz. ketohexokinase, 1-phosphofructokinase and fructose 1,6-bisphosphate aldolase were present under all growth conditions, their levels were elevated in presence of fructose. Besides, though fructose 1,6-bisphosphatase, phosphohexoseisomerase and glucose 6-phosphate dehydrogenase were present, the absence of 6-phosphogluconate dehydratase precluded routing of fructose through ED pathway, or through PP pathway directly as 6-phosphogluconate dehydrogenase was lacking. Fructose 1,6-bisphosphatase plays the unusual role of a catabolic enzyme in supporting the non-oxidative part of PP pathway. However the presence of constitutive levels of glucose dehydrogenase and 2-keto 3-deoxy 6-phosphogluconate aldolase when glucose or sucrose were growth substrates suggested that glucose breakdown took place via the modified ED pathway.Abbreviations EMP Embden Meyerhof Parnas - ED Entner Doudoroff - PP pentose phosphate - KHK ketohexokinase - 1-PFK 1-phosphofructokinase - PEP-PTS phosphoenolpyruvate phosphotransferase - 6-PFK 6-phosphofructokinase - FBPase fructose 1,6-bisphosphatase - PHI phosphohexoseisomerase - G6P-DH glucose 6-phosphate dehydrogenase - 6PG-DH 6-phosphogluconate dehydrogenase - GAPDH glyceraldehyde 3-phosphate dehydrogenase - FIP fructose 1-phosphate - GSH reduced glutathione - 2-ME -mercaptoethanol - FBP fructose 1,6-bisphosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - F6P fructose 6-phosphatez     

Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Sequence, protein homology, and expression during growth on glucose

We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The predicted protein sequence for fructose-1,6-bisphosphatase from S. cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S. pombe, contains 346 amino acids and has a molecular weight of 38,380. Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence. These homologous regions are likely candidates for functional domains. A gene cassette was constructed for fructose-1,6-bisphosphatase from S. cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast. Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions. Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.     

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.     

Chloroplast-Diphenyl Ether Interactions II

Acifluorfen, a p-nitrodiphenyl ether herbicide, is inhibitory to those photosynthetic functions that require a functioning chloroplast envelope. Functions involving the stroma are also affected. Acifluorfen does not lyse intact spinach chloroplasts, yet does increase the sensitivity of CO₂-dependent O₂ evolution to exogenous inorganic phosphate without directly affecting the function of the phosphate translocator. Acifluorfen penetrates into the chloroplast stroma in a light-independent fashion. Once inside, it causes the inactivation of light and dithiothreitol-activated fructose 1,6-bisphosphatase. Light-activated glyceraldehyde-3-phosphate dehydrogenase (NADP) is also inactivated by acifluorfen.

These data suggest that acifluorfen stimulates a pathway for inactivation of fructose 1,6-bisphosphatase and glyceraldehyde 3-phosphate dehydrogenase (NADP) which uses oxygen as a terminal oxidant and which involves thioredoxin and ferredoxin-thioredoxin reductase.