Cerevisiae Phosphoenolpyruvate Carboxykinase Research Articles

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn 2+

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO 2. They are activated by Mn 2+, a metal ion that coordinates to the protein through the ɛ-amino group of a lysine residue, the N ɛ-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ɛ-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ɛ-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn 2+ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn 2+ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn 2+. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.

Lysine 213 and histidine 233 participate in Mn(II) binding and catalysis in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: theoretical and experimental study of the effect of glutamic acid 284 on the protonation state of lysine 213

The crystal structure of Escherichia coli phosphoenolpyruvate (PEP) carboxykinase shows Lys 213 is one of the ligands of enzyme-bound Mn 2+ [Nat. Struct. Biol. 4 (1997) 990]. The direct coordination of Mn 2+ by N ε of Lys 213 is only consistent with a neutral (uncharged) Lys 213, suggesting a low p K a for this residue. This work shows, through theoretical calculations and experimental analyses on homologous Saccharomyces cerevisiae PEP carboxykinase, how the microenvironment affects Mn 2+ binding and the protonation state of Lys 213. We show that Glu 284, a residue close to Lys 212, is required for correct protonation states of Lys 212 and Lys 213, and for Mn 2+ binding. Δ G and Δ H values for the proton reorganization processes were calculated to analyze the energetic stability of the two different protonation states of Lys 212 and Lys 213 in wild-type and Glu284Gln S. cerevisiae PEP carboxykinase. Calculations were done using two modeling approaches, ab-initio density functional calculations and free energy perturbation (FEP) calculations. Both methods suggest that Lys 212 must be protonated and Lys 213 neutral in the wild-type enzyme. On the other hand, the calculations on the Glu284Gln mutant suggest a more stable neutral Lys 212 and protonated Lys 213. Experimental measurements showed 3 orders of magnitude lower activity and a threefold increase in K m for Mn 2+ for Glu284Gln S. cerevisiae PEP carboxykinase when compared to wild type. The data here presented suggest that Glu 284 is required for Mn 2+ binding by S. cerevisiae PEP carboxykinase. We propose that Glu 284 modulates the p K a value of Lys 213 through electrostatic effects mediated by Lys 212.

Urea-induced unfolding studies of free- and ligand-bound tetrameric ATP-dependent Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase : Influence of quaternary structure on protein conformational stability

ATP-dependent phosphoenolpyruvate (PEP) carboxykinases are found in plants and microorganisms, and catalyse the reversible formation of PEP, ADP, and CO 2 from oxaloacetate plus ATP. These enzymes vary in quaternary structure although there is significant sequence identity among the proteins isolated from different sources. To help understand the influence of quaternary structure in protein stability, the urea-induced unfolding of free- and substrate-bound tetrameric Saccharomyces cerevisiae PEP carboxykinase is described and compared with the unfolding characteristics of the monomeric Escherichia coli enzyme [Eur. J. Biochem. 255 (1998) 439]. The urea-induced denaturation of S. cerevisiae PEP carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism (CD) spectra, and 1-anilino-8-naphthalenesulfonate (ANS) binding. The unfolding profiles were multi-steps, and formation of hydrophobic structures were detected. The data indicate that unfolding and dissociation of the enzyme tetramer are simultaneous events. Ligand binding, most notably PEP in the presence of MnCl 2, conferred a marked protection against urea-induced denaturation. A similar protection effect was found when N-iodoacetyl- N′-(5-sulfo-1-napthyl)ethylene diamine (1,5-I-AEDANS) was covalently bound at Cys 365, within the active site region. Refolding experiments indicated that total recovery of tertiary structure was only obtained from samples previously unfolded to less than 30%. In the presence of substrates, complete refolding was achieved from samples originally denatured up to 50%. The unfolding behaviour of S. cerevisiae PEP carboxykinase was found to be similar to that of E. coli PEP carboxykinase, however all steps take place at lower urea concentrations. These findings show that, at least for monomeric and tetrameric ATP-dependent PEP carboxykinases, quaternary structure does not contribute to protein conformational stability.

Molecular modeling of the complexes between Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase and the ATP analogs pyridoxal 5'-diphosphoadenosine and pyridoxal 5'-triphosphoadenosine. Specific labeling of lysine 290.

Molecular mechanics calculations have been employed to obtain models of the complexes between Saccharomyces cerevisiae phosphoenolpyruvate (PEP) kinase and the ATP analogs pyridoxal 5'-diphosphoadenosine (PLP-AMP) and pyridoxal 5'-triphosphoadenosine (PLP-ADP), using the crystalline coordinates of the ATP-pyruvate-Mn(2+)-Mg(2+) complex of Escherichia coli PEP carboxykinase [Tari et al. (1997), Nature Struct. Biol. 4, 990-994]. In these models, the preferred conformation of the pyridoxyl moiety of PLP-ADP and PLP-AMP was established through rotational barrier and simulated annealing procedures. Distances from the carbonyl-C of each analog to epsilon-N of active-site lysyl residues were calculated for the most stable enzyme-analog complex conformation, and it was found that the closest epsilon-N is that from Lys(290), thus predicting Schiff base formation between the corresponding carbonyl and amino groups. This prediction was experimentally verified through chemical modification of S. cerevisiae PEP carboxykinase with PLP-ADP and PLP-AMP. The results here described demonstrate the use of molecular modeling procedures when planning chemical modification of enzyme-active sites.

Interaction of adenosine nucleotide analogs with Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

The substrate characteristics and interactions of different adenosine nucleotide analogs with Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase were investigated by steady-state kinetic analysis and calculations of interaction energies. Comparison of V max/ K m values showed that analogs substituted at C8 in the adenine ring (8-Br-ATP, 8-N 3-ATP, 8-N 3-ADP) gave almost the same kinetic values as ATP and ADP, whereas those substituted in the ribose hydroxyls (3′(2′)- O-( N-methylanthraniloyl)-ATP (MANT-ATP), 3′(2′)- O-( N-methylanthraniloyl)-ADP (MANT-ADP), 2′(3′)- O-(2,4,6-trinitrophenyl)-ADP (TNP-ADP), 2′(3′)- O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP)) showed 1–8% the value for the corresponding physiological substrate. A comparison between the experimental results and molecular mechanics calculations was performed, employing a model for the S. cerevisiae PEP carboxykinase-ATP-Mn 2+ complex. The calculated interaction energies of S. cerevisiae PEP carboxykinase with ATP, MANT-ATP, TNP-ATP, 8-Br-ATP, and 8-N 3-ATP were linearly related (correlation coefficient 0.92) with −ln( V max/ K m). This good correlation supports the proposal that the interaction of the substituent with the enzyme affects the interaction of the common region of ATP with the active site, thus leading to effects in V max.

Circular dichroism and fourier transform infrared spectroscopic studies on the secondary structure of Saccharomyces cerevisiae and Escherichia coli phospho enolpyruvate carboxykinases

The secondary structure of Saccharomyces cerevisiae and Escherichia coli phospho enolpyruvate (PEP) carboxykinases was quantitatively examined using circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies. From CD analyses, values of 24% α-helix and 38% β-sheet were obtained for the E. coli enzyme, while the corresponding values for the S. cerevisiae PEP carboxykinase were 20% and 36%. Analysis of the amide I' infrared band indicated 20% α-helix and 36% β-sheet for the S. cerevisiae enzyme, while for the E. coli protein values of 40% β-sheet and between 9 and 36% α-helix could be inferred. It is concluded that the bacterial enzyme has more secondary structure elements than the yeast protein. No alteration of the CD or FTIR spectra was detected upon substrate or metal ion binding to any enzyme.

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: revised amino acid sequence, site-directed mutagenesis, and microenvironment characteristics of cysteines 365 and 458.

Two cysteine residues in phosphoenolpyruvate (PEP) carboxykinase from Saccharomyces cerevisiae [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] the modification of which leads to enzyme inactivation have been subjected to site-directed mutagenesis. PEP carboxykinase is inactivated by alkylation of Cys365 or Cys458; however, mutation of either or both of these residues to serine has little effect on the enzymatic activity. These results eliminate any possible catalytic function for these cysteinyl residues. In the course of this work, discrepancies in the published nucleotide sequence of the S. cerevisiae PEP carboxykinase gene were detected that alter the deduced amino acid sequence. Several of these discrepancies were verified through the sequencing of proteolytic peptides. Our results indicate that the protein corresponds to a 549 amino acid polypeptide and that the positions previously assigned to Cys364 and Cys457 correspond to Cys365 and Cys458. The individual reactivities and the microenvironment characteristics around these sulfhydryl groups were investigated by their selective modification with the fluorescent reagent N-(1-pyrenyl)maleimide (PyM). Our findings indicate that Cys458 is 7-fold more reactive toward the sulfhydryl-directed probe than Cys365, while quenching experiments of PyM-labeled mutant enzymes suggest that the former residue is located in a region more accessible to water than the latter.

Comparative steady-state fluorescence studies of cytosolic rat liver (GTP), Saccharomyces cerevisiae (ATP) and Escherichia coli (ATP) phospho enolpyruvate carboxykinases

Two members of the ATP-dependent class of phospho enolpyruvate (PEP) carboxykinases ( Saccharomyces cerevisiae and Escherichia coli PEP carboxykinase), and one member of the GTP-dependent class (the cytosolic rat liver enzyme) have been comparatively analyzed by taking advantage of their intrinsic fluorescence. The S. cerevisiae and the rat liver enzymes show intrinsic fluorescence with a maximum emission characteristic of moderately buried tryptophan residues, while the E. coli carboxykinase shows somewhat more average exposure for these fluorophores. The fluorescence of the three proteins was similarly quenched by the polar compound acrylamide, but differences were observed for the ionic quencher iodide. For the ATP-dependent enzymes, these last experiments indicate more exposure to the aqueous media of the tryptophan population of the E. coli than of the S. cerevisiae enzyme. The effect of nucleotides on the emission intensities and quenching efficiencies revealed substrate-induced conformational changes in the E. coli and cytosolic rat liver PEP carboxykinases. The addition of Mn 2+ or of the adenosine nucleotides in the presence of Mg 2+ induced an enhancement in the fluorescence of the E. coli enzyme. The addition of guanosine or inosine nucleotides to the rat liver enzyme quenched its fluorescence. From the ligand-induced fluorescence changes, dissociation constants of 40 ∓ 6 μM, 10 ± 0.8 / gmM, and 15 ± 1 μM were obtained for Mn 2+, MgATP and MgADP binding to the E. coli enzyme, respectively. For the cytosolic rat liver PEP carboxykinase, the respective values for GDP, IDP and ITP binding are 6 ± 0.5 μM, 6.7 ± 0.4 μM and 10.1 ± 1.7 μM. A comparison of the dissociation constants obtained in this work with those reported for other PEP carboxykinases is presented.