Catabolite inactivation of phosphoenolpyruvate carboxykinase in spheroplasts from Saccharomyces Cerevisiae

Catabolite inactivation of phosphoenolpyruvate carboxykinase was studied in yeast spheroplasts using 0.9 M mannitol or 0.6 M potassium chloride as the osmotic support. In the presence of potassium chloride the rate of catabolite inactivation was nearly the same as that occurring in intact yeast cells under different conditions of incubation. However, in the presence of mannitol, catabolite inactivation in spheroplasts was prevented. The mannitol inhibition of catabolite inactivation was released by addition of ammonium or phosphate ions. At a concentration of 0.3 M ammonium or 0.06 M phosphate ions, the maximum rate of catabolite inactivation in spheroplasts suspended in mannitol was achieved and was comparable with that observed in spheroplasts incubated in 0.6 M potassium chloride as the osmotic stabilizer. Sodium sulfate (0.04 and 0.4 M) or potassium chloride (0.06 and 0.6 M) did not release the mannitol inhibition of catabolite inactivation in spheroplasts. In intact yeast cells, 0.9 M mannitol, 0.08 M ammonium or 0.1 M phosphate ions did not influence the rate of catabolite inactivation. The nature of the effects of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is disscussed.     

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.     

Inactivation by glucose of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

Phosphoenolpyruvate carboxykinase showed high activity in Saccharomyces cerevisiae grown on gluconeogenic carbon sources. Addition of glucose to such cultures caused a rapid loss of the phosphoenolpyruvate carboxykinase activity. Fructose or mannose had the same effect as glucose, while 2-deoxyglucose or galactose were without effect. The inactivation was an irreversible process, since the regain of the activity was dependent of de novo protein synthesis. Cycloheximide did not prevent inactivation. All strains of the genus Saccharomyces tested showed inactivation of their phosphoenolpyruvate carboxykinase upon addition of glucose; this behaviour was not restricted to this genus.Non-Standard Abbreviations FbPase fructose bisphosphatase [EC 3.1.3.11 fructose-1,6-bisphosphate hydrolase] - PEPCK phosphoenolpyruvate carboxykinase [EC 4.1.49 ATP: oxalacetate carboxylase (transphosphorylating)] - YPE yeast-peptone-ethanol A preliminary account of these results was presented at the Fourth International Symposium on Yeasts, Vienna, Austria, July 1974     

Reactive sulfhydryl groups in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is inactivated by several thiol- and vicinal dithiol-specific reagents. Titration experiments of the enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) show the presence of reactive monothiol and vicinal dithiol groups, whose modifications lead to enzyme inactivation. The enzyme is also inactivated by N-(1-pyrenyl)iodoacetamide (PyrIAM), with a binding stoichiometry of approx. 2 mol per mol of enzyme subunit. A high level of pyrene excimer fluorescence is detected on the labeled enzyme, thus implying the reaction of the reagent with two spatially close sulfhydryl groups in the protein. The carboxykinase is not completely inactivated by different vicinal dithiol-specific reagents, thus implying a catalytically non-essential character for these groups. From substrate protection experiments of the enzyme inactivation by DTNB, PyrIAM and vicinal dithiol-specific reagents, it is concluded that the loss of enzyme activity is caused by the modification of both thiol and vicinal dithiol groups in the substrate binding region.     

The presence of functional arginine residues in phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is completely inactivated by phenylglyoxal and 2,3-butanedione in borate buffer at pH 8.4, with pseudo-first-order kinetics and a second-order rate constant of 144 min-1 X M-1 and 21.6 min-1 X M-1, respectively. Phosphoenolpyruvate, ADP and Mn2+ (alone or in combination) protect the enzyme against inactivation, suggesting that the modification occurs at or near to the substrate-binding site. Almost complete restoration of activity was obtained when a sample of 2,3-butanedione-inactivated enzyme was freed of excess modifier and borate ions, suggesting that only arginyl groups are modified. The changes in the rate of inactivation in the presence of substrates and Mn2+ were used to determine the dissociation constants for enzyme-ligand complexes, and values of 23 +/- 3 microM, 168 +/- 44 microM and 244 +/- 54 microM were found for the dissociation constants for the enzyme-Mn2+, enzyme-ADP and enzyme-phosphoenolpyruvate complexes, respectively. Based on kinetic data, it is shown that 1 mol of reagent must combine per enzyme active unit in order to inactivate the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of 3-4 mol [7-14C]phenylglyoxal per mol of enzyme subunit. Assuming a stoichiometry of 1:1 between phenylglyoxal incorporation and arginine modification, our results suggest that the modification of only two of the three to four reactive arginine residues per phosphoenolpyruvate carboxykinase subunit is responsible for inactivation.     

Studies on the degradative mechanism of phosphoenolpyruvate carboxykinase from yeast Saccharomyces cerevisiae

Previous work carried out in our laboratory (Burlini, N., Lamponi S., Radrizzani, M., Monti, E. and Tortora P. (1987) Biochim. Biophys. Acta 930, 220-229) led to the immunological identification of a yeast 65-kDa phosphoprotein as a modified form of phosphoenolpyruvate carboxykinase; moreover the appearance of this phospho form was proven to be independent of cAMP, whereas the glucose-induced inactivation of the native enzyme is cAMP-dependent. Here, we report further investigations on the mechanism of the glucose-triggered degradation of the enzyme which led to the following results: (a) the aforementioned phospho form displayed a binding pattern to 5 AMP-Sepharose 4B quite similar to that of native enzyme, although it did not retain its oligomeric structure, nor was it catalytically active; (b) its phosphate content was of about two residues per monomer; (c) its isoelectric point was slightly higher than that of native enzyme, this shows that the enzyme undergoes additional modifications besides phosphorylation; (d) it represented about 4% of the native enzyme in glucose-depressed cells; (e) other forms immunologically cross-reactive with the native enzyme were also isolated, whose molecular mass was in the range of 60-62 kDa, and they are probable candidates as degradation products of the phospho form; (f) time courses of the native and phospho forms in the presence and the absence of glucose provided data consistent with a kinetic model involving a strong stimulation of the decay of both forms effected by the sugar; (g) in the mutant ABYS1 (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343) which is devoid of the four major vacuolar proteinases, the decay pattern was essentially the same as in wild-type; (h) effectors lowering intracellular ATP also retarded the first step of enzyme degradation; this points to an ATP-dependence of this step. Based on these results we propose a degradation mechanism consisting of an initial cAMP- and ATP-dependent modification of the enzyme, followed by a cAMP-independent phosphorylation, which leads to the appearance of the aforementioned monomeric phospho form; this in turn seems to undergo limited proteolysis. These data strongly suggest the occurrence of an intermediate form arising from the native one and whose phosphorylation gives rise to the 65-kDa phosphoprotein described here.     

Isolation and characterization of the gene encoding phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

The yeast PCK1 gene coding for phosphoenolpyruvate carboxykinase (PEPCK) was isolated by functional complementation of pck1 strains from S. cerevisiae. Only one copy of the gene was found per haploid yeast genome. An RNA of about 2 kb which hybridized with a DNA probe internal to the PCK1 gene was found only in cells growing in non-fermentable carbon sources. Yeast strains carrying multiple copies of the PCK1 gene showed normal catabolite repression of PEPCK except those carrying the shortest insertion complementing the mutation (2.2 kb) that presented an altered kinetics of derepression. Catabolite inactivation was decreased in strains transformed with multicopy plasmids carrying the PCK1 gene.     

Functional evaluation of serine 252 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase mutant Ser252Ala, affecting the conserved Walker A serine residue, was characterized to elucidate the role of this serine residue. The substitution did not result in changes in the protein structure, as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analysis of the mutated enzyme in both directions of the main reaction and in the two secondary reactions showed an approximately 50-fold increase in apparent K(m) for oxaloacetate with minor alterations in the other kinetic parameters. These results show that the hydroxyl group of serine 252 is required for proper oxaloacetate interaction.     

Catabolite inactivation of the glucose transport system in Saccharomyces cerevisiae

The sugar transport systems of Saccharomyces cerevisiae are irreversibly inactivated when protein synthesis is inhibited. This inactivation is responsible for the drastic decrease in fermentation observed in ammonium-starved yeast and is related to the occurrence of the Pasteur effect in these cells. Our study of the inactivation of the glucose transport system indicates that both the high-affinity and the low-affinity components of this system are inactivated. Inactivation of the high-affinity component evidently requires the utilization of a fermentable substrate by the cells, since inactivation did not occur during carbon starvation, when a fermentable sugar was added to starved cells, inactivation began, when the fermentation inhibitors iodoacetate or arsenate were added in addition to sugars, the inactivation was prevented, when a non-fermentable substrate was added instead of sugars, inactivation was also prevented. The inactivation of the low-affinity component appeared to show similar requirements. It is concluded that the glucose transport system in S. cerevisiae is regulated by a catabolite-inactivation process.     

Identification of a phosphorylated form of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae

A phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid.     

Expression of the Trypanosoma brucei phosphoenolpyruvate carboxykinase gene in Saccharomyces cerevisiae

Plasmid pTbp60B (Kueng et al., J. Biol. Chem. 264 (1989) 5203-5209) was employed to obtain, through the polymerase chain reaction, the Trypanosoma brucei gene coding for phosphoenolpyruvate (PEP) carboxykinase, and then cloned into the yeast expression plasmid pYES2. The cloned gene was completely sequenced and the expression plasmid transformed into Saccharomyces cerevisiae PUK-3B (MATalpha pck1 ura3 ade1) competent cells. Gene expression took place upon induction with 2% galactose, and the recombinant T. brucei PEP carboxykinase was purified to near homogeneity. The basic molecular and catalytic characteristics of the recombinant enzyme were determined, and they showed to be essentially similar to those reported for wild type T. brucei PEP carboxykinase (Hunt and K?hler, Biochim. Biophys. Acta 1249 (1995) 15-22). The expression system here described is a reliable non-pathogenic source of T. brucei PEP carboxykinase.     

Purification of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae and its use for bicarbonate assay

Electrophoretically homogeneous phosphoenolpyruvate carboxykinase (EC 4.1.1.49) from Saccharomyces cerevisiae was obtained in high yields by means of a two-step purification procedure consisting of ion-exchange chromatography and affinity chromatography on adenosine 5'-monophosphate-Sepharose 4B. In the latter step the binding of the enzyme to the resin specifically required the presence of Mn2+. The enzyme was eluted when Mn2+ was removed by addition of ethylenediaminetetraacetate to the elution buffer. Homogeneity, molecular weight, and subunit composition of phosphoenolpyruvate carboxykinase were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. A factor which caused an underestimation of the enzyme activity in crude extracts was identified as adenylate kinase. Finally, a method is proposed for the enzymatic assay of bicarbonate using a purified phosphoenolpyruvate carboxykinase preparation.     

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: relevance of arginine 70 for catalysis

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase is a key enzyme of the gluconeogenic pathway and catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2 in the presence of a divalent metal ion. Previous experiments indicate that mutation of amino acid residues at metal site 1 decrease the enzyme catalytic efficiency and the affinity of the protein for PEP, evidencing the relevance of hydrogen-bond interactions between PEP and water molecules of the first coordination sphere of the metal ion for catalysis [Biochemistry 41 (2002) 12763]. To further understand the function of amino acid residues located in the PEP binding site, we have now addressed the catalytic importance of Arg70, whose guanidinium group is close to the PEP carboxyl group. Arg70 mutants of PEP carboxykinase were prepared, and almost unaltered kinetic parameters were found for the Arg70Lys PEP carboxykinase, while a decrease in 4-5 orders of magnitude for the catalytic efficiency was detected for the Arg70Gln and Arg70Met altered enzymes. To evaluate the enzyme interaction with PEP, the phosphopyridoxyl-derivatives of wild type, Arg70Lys, Arg70Gln, and Arg70Met S. cerevisiae PEP carboxykinase were prepared, and the change in the fluorescence emission of the probe upon PEP binding was used to obtain the dissociation equilibrium constant of the corresponding derivatized enzyme-PEP-Mn2+ complex. The titration experiments showed that a loss in 2.1 kcal/mol in PEP binding affinity is produced in the Arg70Met and Arg70Gln mutant enzymes. It is proposed that the electrostatic interaction between the guanidinium group of Arg70 and the carboxyl group of PEP is important for PEP binding and for further steps in catalysis.     

Isolation and characterization of a mutant of Saccharomyces cerevisiae defective in phosphoenolpyruvate carboxykinase

A mutant of Saccharomyces cerevisiae lacking phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) was isolated. The mutant did not grow on gluconeogenic sources except glycerol. The mutation was recessive and apparently affected the structural gene of the enzyme. Intracellular levels of metabolites related to the metabolic situation of the enzyme were not significantly affected after transfer of the mutant from a medium with glycerol to a medium with ethanol as carbon source. In these conditions only AMP decreased 3 to 5 times. A search for mutants affected in the other gluconeogenic enzyme, fructose 1,6 bisphosphatase, remained unsuccessful.Abbreviation PEPCK phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32)     

Limited proteolysis ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Incubation ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase with trypsin under native conditions cases a time-dependent loss of activity and the production of protein fragments. Cleavage sites determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and sequence analyses identified protease-sensitive peptide bonds between amino acid residues at positions 9–10 and 76–77. Additional fragmentation sites were also detected in a region approximately 70–80 amino acids before the carboxyl end of the protein. These results suggest that the enzyme is formed by a central compact domain comprising more than two thirds of the whole protein structure. From proteolysis experiments carried out in the presence of substrates, it could be inferred that CO₂ binding specifically protects position 76–77 from trypsin action. Intrinsic fluorescence measurements demonstrated that CO₂ binding induces a protein conformational change, and a dissociation constant for the enzyme CO₂ complex of 8.2±0.6 mM was determined     

Relevance of Arg457 for the nucleotide affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Phosphoenolpyruvate carboxykinases catalyze one of the first steps in the biosynthesis of glucose and depending on the enzyme origin, preferentially use adenine or guanine nucleotides as substrates. The Saccharomyces cerevisiae enzyme has a marked preference for ADP (or ATP) over other nucleotides. Homology models of the enzyme in complex with ADP or ATP show that the guanidinium group of Arg457 is close to the adenine base, suggesting that this group might be involved in the stabilization of the nucleotide substrate. To evaluate this we have performed the mutation Arg457Met, replacing the positively charged guanidinium group by a neutral residue. The mutated enzyme retained the structural characteristics of the wild-type protein. Fluorescence titration experiments showed that mutation causes a loss of 1.7 kcal mol(-1) in the binding affinity of the enzyme for ADPMn. Similarly, kinetic analyses of the mutated enzyme showed 50-fold increase in K(m) for ADPMn, with minor alterations in the other kinetic parameters. These results show that Arg457 is an important factor for nucleotide binding by S. cerevisiae PEP carboxykinase.     

The role of tyrosine 207 in the reaction catalyzed by Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

The functional significance of tyrosine 207 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase was explored by examining the kinetic properties of the Tyr207Leu mutant. The variant enzyme retained the structural characteristics of the wild-type protein as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analyses of the mutated variant showed a 15-fold increase in K(m)CO?, a 32-fold decrease in V(max), and a 6-fold decrease in K(m) for phosphoenolpyruvate. These results suggest that the hydroxyl group of Tyr 207 may polarize CO? and oxaloacetate, thus facilitating the carboxylation/decarboxylation steps.     

Activation and inactivation of phosphoenolpyruvate carboxykinase by ferrous ions.

Phosphoenolpyruvate carboxykinase from rat liver cytosol is activated by Fe2+ ions in either direction of catalysis. Preincubation of the purified enzyme with Fe2+ ions causes a time-dependent irreversible loss of activity; this is not seen with unpurified enzyme. Purified enzyme can be protected from inactivation by Fe2+ ions by partially purified protein fractions from liver (ferroactivator fractions). The possible role of ferroactivator and Fe2+ ions in regulating phosphoenolpyruvate carboxykinase is discussed.     

Osmotic imbalance in inositol-starved spheroplasts of Saccharomyces cerevisiae.

Physiological states associated with inositol starvation of spheroplasts of Saccharomyces cerevisiae were investigated and compared with conditions preceding death of starved whole cells. In the absence of synthesis of inositol-containing lipids, cell surface expansion terminated after one doubling of whole cells. In spheroplasts, cessation of membrane expansion was apparently followed by rapid development of an osmotic imbalance, causing lysis. Continued synthesis and accumulation of cytoplasmic constituents within the limited cell volume were implicated as a cause of the osmotic imbalance. In whole cells, an increase in internal osmotic pressure also follows termination of membrane and cell wall expansion. The cell wall prevents lysis, allowing a state of increasing cytoplasmic osmotic pressure to persist in the period preceding onset of inositol-less death.