Time resolved spectroscgpy of tryptopkyl fluorescence of yeast 3-phosphoglycerate kinase

The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime τ₁= 0.6 ns and a maximum fluorescence wavelength λ_2max = 332 nm. The second tryptophan residue exhibits two lifetimes τ₂₁= 3.1 ns and τ₂₂= 7.0 ns (λ_2max= 338 nm). In agreement with the attribution of τ₂₁and τ₃₂ to the same tryptophan residue, the ratio β = C₂₁/C₂₂ of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally tc the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay times and a neighbouring protein chemical group. The intrinsic pK of this group and the equilibrium constant of the irternal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.     

Substrate-induced conformational changes in yeast 3-phosphoglycerate kinase monitored by fluorescence of single tryptophan probes.

3-Phosphoglycerate kinase (PGK) catalyzes the reversible conversion of 3-phosphoglycerate (3-PG) and ATP to 1,3-diphosphoglycerate (1,3-diPG) and ADP in the presence of magnesium ions. PGK is a single polypeptide chain arranged in two domains, with an active site located in the interdomain cleft. The large distance between the binding sites for 3-PG and ATP, deduced from the crystallographic structures of the binary complexes, gave rise to the hypothesis that this enzyme undergoes a hinge-bending domain motion from open to closed conformation during catalysis. However, no direct experimental evidence exists for the "closed" conformation in the presence of both substrates. In this study, several PGK mutants with single tryptophans placed in various location were used as intrinsic fluorescent probes to examine the extent and delocalization of conformational changes induced by the binding of 3-PG, 1,3-diPG, ADP, ATP, and PNP-AMP (nonhydrolyzable analogue of ATP), and by 3-PG and PNP-AMP together. The results showed that only the probes situated in the hinge and in parts of each domain close to the hinge reflect substrate-induced conformational changes. Binding of substrates to one domain was found to induce spectral perturbation of the probes in the opposite domain, indicating a transmission of conformational changes between the domains. A combination of both substrates generated much larger fluorescence changes than the individual substrates. The binding constants were determined for each substrate using probes situated in different locations.     

Resolution of the fluorescence of the buried tryptophan in yeast 3-phosphoglycerate kinase using succinimide

The heterogeneous fluorescence of yeast 3-phosphoglycerate kinase, a hinge-bending enzyme with two tryptophans, has been resolved into two approximately equal components, one accessible and one inaccessible to the relatively inefficient quencher succinimide. The inaccessible component is blue-shifted and exhibits a heterogeneous fluorescence decay which has a temperature-dependence and steady-state acrylamide quenching properties typical of a single tryptophan in a buried environment. This component is therefore assigned to the buried tryptophan W333. The presence of succinimide greatly simplifies the fluorescence allowing the conformational dynamics of the buried tryptophan and its environment to be studied without interference from the other tryptophan.     

Human, yeast and hybrid 3-phosphoglycerate kinase gene expression in yeast.

When the gene for yeast 3-phosphoglycerate kinase (PGK) is present on a high copy number plasmid in Saccharomyces cerevisiae, 30-40 percent of yeast protein is produced as PGK. However, when the structural part of this gene is replaced by as many as twenty different heterologous genes, production of gene products is greatly reduced--usually by more than 20 fold. This decrease in protein production is accompanied by large decreases in the steady-state levels of mRNA. However, in contrast to these coding sequences, replacement of the yeast PGK structural gene with a human PGK cDNA has little effect on the steady-state mRNA level in yeast. PGK is a two-domain enzyme and its 3-dimensional structure is highly conserved among species. These observations and others have led us to propose that the PGK protein itself might influence its own mRNA levels (Chen et al., Nucleic Acids Res. 12, pp. 8951-8969, 1984). In addition, data is presented here which suggest that the human PGK mRNA is less efficiently translated than the yeast PGK mRNA. Two different mechanisms of controlling gene expression are indicated. Both mechanisms appear to be independent of gene copy number.     

The steady-state kinetics of yeast phosphoglycerate kinase. Anomalous kinetic plots and the effects of salts on activity.

1. A re-investigation of the kinetics of yeast phosphoglycerate kinase in the direction of 1,3-bisphosphoglycerate formation has been carried out, covering a 1000-fold range in substrate concentrations. A variety of improved spectrophotometric and fluorimetric assay procedures have been used. 2. Kinetic plots proved to be non-linear for each variable substrate. A variety of checks have been carried out to show that this is not due to artifacts in the assay procedures or heterogeneity of the enzyme preparation. 3. The effects of a variety of salts on the activity of the enzyme have been examined. Most salts, especially those with multivalent anions, can cause activation of the enzyme, but inhibit at high concentration. 4. The salt effect is shown to be principally due to anions rather than cations, and not to ionic strength changes. Sulphate, as one of the most effective anions has been used in most comparisons. 5. Salt activation is steepest when the substrate concentrations are low; maximum activation has been about 5-fold with 0.2 mM MgATP and 0.2 mM 3-phosphoglycerate. Inhibition at the higher salt concentrations is strongest at the same substrate concentrations as when activation is steepest, indicating a link between the two effects. 6. The presence of 20 mM or more Na2SO4 converted non-linear kinetic plots to linear ones. A study of the kinetics in the presence of 40 mM Na2SO4 was interpreted in terms of a random sequential binding mechanism, with sulphate acting as a competitive inhibitor. 7. Possible explanations for these anomalous results are discussed in terms of several mechanisms which have been shown to apply in other systems.     

Protamine kinase from yeast.

A protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) which preferentially phosphorylates protamine is purified about 250-fold from the soluble fraction of baker's yeast (Saccharomyces cerevisiae). This enzyme is not sensitive to activation by cyclic nucleotides. Histone is about 5% as active as protamine in the reaction rate. Neither casein, phosvitin nor glycogen phosphorylase is active as substrate. The enzyme is distinguishable from casein kinase of the classical type (Rabinowitz, M. and Lipmann, F. (1960) J. Biol. Chem. 235, 1043-1050) and from adenoshine 3', 5'-monophosphate-dependent protein kinase described earlier (Takai, Y., Yamamura, H. and Nishizuka, Y. (1974) J. Biol. Chem. 249,530-535).     

Frequency domain fluorescence studies of yeast phosphoglycerate kinase and its ternary complex

A frequency domain fluorescence study of yeast phosphoglycerate kinase has been performed to observe the effect of substrates on the structure and dynamics of the enzyme. At 20 degrees C and pH 7.2, a biexponential decay is observed for tryptophanyl emission. The short fluorescence lifetime (0.4 ns) component is associated with a spectrum having a 329-nm maximum and a 18.4-kJ/mol activation energy, Ea, for thermal quenching. The long-lifetime (3.5 ns) component has a 338-nm maximum and an Ea of only 7.9 kJ/mol. Tentatively we assign the short and long-lifetime components to Trp-333 and Trp-308. Binding of the substrates ATP and 3-phosphoglycerate leads to a significant increase in the fluorescence lifetime, the red shift of the emission spectrum and in the decrease in the Ea for both components. Acrylamide-quenching studies indicate that the two tryptophan residues have about the same degree of kinetic exposure to the quencher and that the binding of the substrates causes a very slight change in the quenching pattern. These fluorescence studies indicate that the binding of the substrates to phosphoglycerate kinase may influence the conformational dynamics around the two tryptophan residues located on one of the protein's domains.     

A study of the hinge-bending mechanism of yeast 3-phosphoglycerate kinase.

The hinge-bending mechanism proposed as part of the catalytic mechanism for phosphoglycerate kinase (PGK) has been investigated using yeast PGK and the site-directed mutant [H388Q]PGK, where His388 is replaced by Gln. The emission and quenching of fluorescence, supported by the aromatic CD band, show that the mutation in the waist region affects the tryptophan environment in the C-terminal domain. The mutant is also less stable to guanidine denaturation and less cooperative in its unfolding. The effect of substrates on the conformation of PGK was studied using 8-anilino-1-naphthalenesulphonic acid (ANS), a competitive inhibitor of ATP binding to the C-terminal domain, and 8-(2-[(iodoacetyl)ethyl]amino)naphthalene (I-AEDANS), attached to Cys197 on the N-terminal domain. Under the influence of substrates the novel anisotropy decay curves for ANS indicate a 1-5 degrees change in the orientation of the probe, interpreted as a small reorientation of the domains about the waist region. The experimental data are interpreted as a small swivelling of the domains about the waist region under the influence of substrate. The results with AEDANS anisotropy decay are consistent with those for ANS. The enzyme activity of PGK shows a break in the Arrhenius plot at 20 degrees C mirrored by a break in the temperature dependence of tryptophan ellipticity. This is interpreted as a change in protein dynamics associated with destabilisation of the waist region. This destabilisation is shown to have already taken place in the mutant enzyme and in the wild type at pH 5.6, both of which exhibit linear Arrhenius plots. NMR titration curves show that the pH effect must be due to a group other than histidine. The results give further support to the permissive model of hinge bending previously proposed by one of the authors, in which binding of substrate destabilises the waist region. This loosens the hinge which can then swing slightly to bring the domains closer together to make favourable interactions between the domains and the substrates, with the exclusion of water.     

Secondary kinase reactions catalyzed by yeast pyruvate kinase.

1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. Wth Mg2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-biphosphate. With Mn2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on three secondary kinase reactions was also examined. 4. Results are compared with those obtained from muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.     

MAP kinase dynamics in yeast.

MAP kinase pathways play key roles in cellular responses towards extracellular signals. In several cases, the three core kinases interact with a scaffold molecule, but the function of these scaffolds is poorly understood. They have been proposed to contribute to signal specificity, signal amplification, or subcellular localization of MAP kinases. Several MAP kinases translocate to the nucleus in response to their activation, suggesting that nuclear transport may provide a regulatory mechanism. Here we describe new applications for Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP), to study dynamic translocations of MAPKs between different subcellular compartments. We have used these methods to measure the nuclear/cytoplasmic dynamics of several yeast MAP kinases, and in particular to address the role of scaffold proteins for MAP-kinase signaling.     

Anion binding to yeast phosphoglycerate kinase.

The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2-chloromercuri-4-nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from--1 to--4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme-anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as Cl-. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000 g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored. Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5'-dithiobis(2-nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme-mercurial complex.     

The regulatory properties of yeast pyruvate kinase.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 where the relationships between the initial velocities of the catalysed reaction and the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The findings were represented empirically by the exponential model for a regulatory enzyme. The analysis shows that ADP, phosphoenolpyruvate and Mg2+ display positive homotropic interaction in their binding behaviour with (calculated) Hill slopes at half-saturation equal to 1.06, 2.35 and 3.11 respectively [Ainsworth (1977) J. Theor. Biol. 68, 391-413]. The direct heterotropic interaction between ADP and phosphoenolpyruvate is small and negative, but the overall interaction between these substrates becomes positive when their positive interactions with Mg2+ are taken into account. The heterotropic interactions of the substrates, though smaller in magnitude, are comparable with those revealed by the rabbit muscle enzyme [Ainsworth, Kinderlerer & Gregory (1983) Biochem. J. 209, 401-411], and it is suggested that they have a common origin in charge interactions within the active site.     

Sequence and structure of yeast phosphoglycerate kinase.

The structure of yeast phosphoglycerate kinase has been determined with data obtained from amino acid sequence, nucleotide sequence, and X-ray crystallographic studies. The substrate binding sites, as deduced from electron density maps, are compatible with known substrate specificity and the stereochemical requirements for the enzymic reaction. A carboxyl-imidazole interaction appears to be involved in controlling the transition between the open and closed forms of the enzyme.     

Stimulation of yeast 3-phosphoglycerate kinase gene promoter by paraquat

Yeast cells exposed to adverse conditions employ a number of defense mechanisms in order to respond effectively to the stress and sustain a high proliferation rate. It has been shown that several glycolytic enzymes are induced upon heat treatment of yeast. In this work, we used a reporter plasmid construct to study the effects of oxidative stress, induced by the O(*-)(2)-generating compound paraquat (PQ), on the yeast 3-phosphoglycerate kinase gene (PGK) promoter. Our results show that (i) moderate, as opposed to excessive, doses of PQ induce increased stimulation of the PGK promoter, at midlogarithmic phase of growth; and (ii) the thiol antioxidant N-acetylcysteine cancels this stimulatory effect. These observations may represent one aspect of a more general role for glycolysis in maintaining the energy pools of yeast cells under stress.     

Interaction of yeast 3-phosphoglycerate kinase with negatively charged carriers

The aim of this study was to investigate the possibility of an interaction of yeast 3-phosphoglycerate kinase with negatively charged carriers such as polyanionic agents or a polarized electrode. Various polyanions were found to promote enzyme aggregation as judged by ultracentrifugation measurements and chemical modification. The data obtained suggest that these interactions are mediated through the N-terminal domain of the protein. However, the most striking property of 3-phosphoglycerate kinase described here is concerned with its significant dipolar moment as evidenced by electrocapillary measurements, which allows an orientation of the macromolecule in an electric field. Further, the enzyme could be absorbed by a negatively charged surface, first by hydrophobic links and then oriented perpendicularly to the surface. Therefore, the intrinsic properties of yeast 3-phosphoglycerate kinase agree with the formation of an enzyme-membrane complex and afford the ability for a specific orientation of the molecule at the lipid bilayer surface or in the cytoplasm.     

Anomalous temperature fluorescence quenching of N-Trp terminal peptides

The photophysics of Trp-containing peptides is extremely affected by the position of the indole ring with respect to the substituents. In this work an unusual temperature fluorescence quenching behavior is presented. The N-tryptophan terminal peptides (N- Trp) show an increase of the static emission intensity as rising the temperature from 10 to about 40°C. The anomaly is typical of the N-Trp terminal peptides since neither tryptophan (Trp) nor glycyl-tryptophan (Gly-Trp) and alanyl-tryptophan (Ala-Trp) show the same trend; a similar behavior is not detected in the C-tryptophan terminals. The other important features are the wavelength and pH dependence of the effect. The anomaly is in fact detected only at neutral pH and for excitation wavelength near the red edge of the UVB absorption band of indole. An interpretation of the anomaly is suggested, though more sophisticated techniques are needed to better focus the problem; the model proposed involves the superimposition of a ground state effect (the temperature-induced equilibrium shift from the zwitterionic to the anionic form of the peptides) and an excited state mechanism. At present no unique interpretation can be provided about the excited state mechanism that favors the anomaly and some suggestions are discussed. © 1995 John Wiley & Sons, Inc.