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.     

Biosynthesis and regulation of fructose-1,6-bisphosphatase and phosphofructokinase in Saccharomyces cerevisiae grown in the presence of glucose and gluconeogenic carbon sources.

The mode of synthesis and the regulation of fructose-1,6-bisphosphatase (Fbpase), a gluconeogenic enzyme, and phosphofructokinase (PFK), a glycolytic enzyme, were investigated in Saccharomyces cerevisiae after growth in the presence of different concentrations of glucose or various gluconeogenic carbon sources. The activity of FBPase appeared in the cells after the complete disappearance of glucose from the growth medium with a concomitant increase of the pH and no significant change in the levels of accumulated ethanol. The appearance of FBPase activity following glucose depletion was dependent upon the synthesis of protein. The FBPase PFK were present in glucose-, ethanol-, glycerol-, lactate-, or pyruvate-grown cells; however, the time of appearance and the levels of both these enzymes varied. The FBPase activity was always higher in 1% glucose-grown cells than in cells grown in the presence of gluconeogenic carbon sources. Phosphoglucose isomerase activity did not vary significantly. Addition of glucose to an FBPase and PFK synthesizing culture resulted in a complete loss, followed by a reappearance, of PFK activity. In the presence of cycloheximide the disappearance of glucose and the changes in the levels of FBPase and PFK were decreased significantly. It is concluded that S. cerevisiae exhibits a more efficient synthesis of FBPase after the exhaustion of glucose compared to the activity present in cells grown in the presence of exogenous gluconeogenic carbon sources. Two metabolically antagonistic enzymes, FBPase and PFK, are present during the transition phase, but not during the exponential phase, of growth, and the decay or inactivation of these enzymes in vivo may be dependent upon a glucose-induced protease activity.     

Gluconeogenesis in Saccharomyces cerevisiae: determination of fructose-1,6-bisphosphatase activity in cells grown in the presence of glycolytic carbon sources.

The activity of fructose-1,6-bisphosphatase (FBP), a gluconeogenic enzyme, was determined in wild-type Saccharomyces cerevisiae X2180 grown in the presence of the glycolytic carbon sources, glucose, fructose, and galactose. The activities of phosphofructokinase (PFK), a glycolytic enzyme, and phosphoglucose isomerase (PGI), an enzyme functioning both in glycolysis and gluconeogenesis, were determined for purposes of comparison. A measurable amount of FBP activity was present in 20-h-old cells grown with moderate shaking in 1% glucose-nutrient or minimal medium. This activity increased significantly in 40 and 60-h-old cells. Similar levels of FBP activity were also present in 20-, 40-, and 60-h-old cells grown in 1% fructose-nutrient medium. A higher level of FBP activity was present in 20-h-old cells grown in 1% galactose-nutrient medium than in 20-h-old cells grown in 1% glucose- or fructose-nutrient medium. The FBP activity in glucose- or fructose-grown cells was higher than the corresponding activity in cells grown under similar conditions for 40 and 60 h in the presence of ethanol, a gluconeogenic carbon source. The PFK activity was significantly less in galactose- and ethanol-grown cells. The PGI activity was relatively constant in 20-, 40-, and 60-h-old cells grown in the presence of glucose, fructose, and galactose, but this activity was reduced approximately 50% in ethanol-grown cells. It is concluded from these results that, depending upon the concentration of carbon source and the time of incubation, FBP, a strictly gloconeogenic enzyme, is synthesized by S. cerevisiae grown in the presence of glycolytic carbon sources.     

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.     

Regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase activity in adult Fasciola hepatica (Trematoda).

F. hepatica pyruvate kinase and phosphoenolpyruvate (PEP) carboxykinase were found to have properties of regulatory enzymes in the dissimilation of PEP and the control of metabolic flow. Mn²⁺ and K⁺ were required for pyruvate kinase activity. In the presence of fructose-1, 6-diphosphate (FDP), Mg²⁺ could substitute for Mn²⁺. FDP caused a 4-fold increase in the Mn²⁺ activated pyruvate kinase activity. This was accompanied by a 12-fold decrease in apparent K_m(PEP) and a 3-fold decrease in apparent K_{m (ADP)}. ATP markedly inhibited F. hepatica pyruvate kinase, but this inhibition was relieved by FDP. Estimates of metabolic levels indicated that the pyruvate kinase is saturated with PEP and ADP in vivo, but will be highly sensitive to fluctuations in the physiological concentrations of FDP and ATP. NADH doubled the activity of the PEP carboxykinase reaction and decreased the apparent K_{m (PEP)} for this enzyme 3-fold. While the maximal activity of the PEP carboxykinase reaction was substantially higher than the pyruvate kinase reaction, the steady state concentration of PEP suggests that the PEP carboxykinase will not be saturated with this substrate.     

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.     

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.     

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.     

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 effect of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is discussed.     

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.     

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)     

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.     

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.     

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.     

Mapping of the PCK1 gene encoding phosphoenolpyruvate carboxykinase on chromosome XI of Saccharomyces cerevisiae

The gene PCK1 encoding phosphoenolpyruvate carboxykinase of Saccharomyces cerevisiae has been mapped on the right arm of chromosome XI, 12.7 centimorgans proximal to MAL4 and 20.1 centimorgans distal to MET1. In order to map the gene, hybridization of PCK1 DNA with separated yeast chromosomes and tetrad analysis of diploids with adequate markers were carried out.     

Identification and characterization of regulatory elements in the phosphoenolpyruvate carboxykinase gene PCK1 of Saccharomyces cerevisiae

Phosphoenolpyruvate carboxykinase is a key enzyme in gluconeogenesis. The expression of the PCK1 gene in Saccharomyces cerevisiae is strictly regulated and dependent on the carbon source provided. Two upstream activation sites (UAS1_PCK1 and UAS2_PCK1) and one upstream repression site (URS_PCK1) were localized by detailed deletion analysis. The efficacy of these three promoter elements when separated from each other was confirmed by investigations using heterologous promoter test plasmids. Activation mediated by UAS1_PCK1 or UAS2_PCK1 did not occur in the presence of glucose, indicating that these elements are essential for glucose derepression. The repressing effect caused by URS_PCK1 was much stronger in glucose-grown cells than in ethanol-grown cells.