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1.
Substrate-induced conformational changes in yeast 3-phosphoglycerate kinase monitored by fluorescence of single tryptophan probes.
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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. 相似文献
2.
The heterogeneous fluorescence of yeast 3-phosphoglycerate kinase, a hinge-bending enzyme with two tryptophan residues, has been resolved into three emission components using steady-state and time-resolved studies of the fluorescence quenching by acrylamide, iodide and caesium ions at different emission wavelengths. The buried Trp-333 has a blue-shifted heterogeneous emission spectrum characterised by three fluorescence lifetimes, and is inaccessible to quenchers. The surface Trp-308 also has a heterogeneous emission with multiple lifetimes. The emission of Trp-308 can be separated into a blue-shifted emission accessible to acrylamide and caesium only, and a red-shifted emission accessible to all three quenchers. 相似文献
3.
Jean-Paul Privat Philippe Wahl Jean-Claude Auchet Roger H. Pain 《Biophysical chemistry》1980,11(2):239-248
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 λ2max = 332 nm. The second tryptophan residue exhibits two lifetimes τ21= 3.1 ns and τ22= 7.0 ns (λ2max= 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. 相似文献
4.
Vassallo N Galea DR Bannister WH Balzan R 《Biochemical and biophysical research communications》2000,270(3):1036-1040
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. 相似文献
5.
C Roustan A Fattoum R Jeanneau L A Pradel D Schuhmann P Vanel 《Biophysical chemistry》1982,15(2):169-176
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. 相似文献
6.
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. 相似文献
7.
R A Stinson 《Biochemistry》1974,13(22):4523-4529
8.
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. 相似文献
9.
An improved procedure for the isolation of 3-phosphoglycerate kinase from yeast 总被引:7,自引:5,他引:2
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R. K. Scopes 《The Biochemical journal》1971,122(1):89-92
1. (+)-gamma-Carboxymethyl-gamma-methyl-Delta(alpha)-butenolide was isolated from resting-cell cultures of Pseudomonas desmolyticum incubated in the presence of 5-methylsalicylic acid. 2. The structure of this metabolite was deduced from physical and chemical evidence. 3. The isolated compound must be formed in an enzymic reaction since it shows optical activity. 4. The degradative pathway of 4-methylcatechol by Ps. desmolyticum is discussed. 相似文献
10.
11.
The folding and mutual interaction of the domains of yeast 3-phosphoglycerate kinase 总被引:2,自引:0,他引:2
Analysis of the reversible unfolding of yeast phosphoglycerate kinase leads to the conclusion that the two lobes are capable of folding independently, consistent with the presence of intermediates on the folding pathway with a single domain folded. The domains have different free energies of stabilisation. Immunological cross-reactivity, circular dichroism and thiol reactivity provide evidence for cyanogen bromide peptide 1-173, which comprises five-sixths of the N-terminal domain, containing sufficient information to refold into a native-like structure which dimerises. 相似文献
12.
Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants. 总被引:4,自引:1,他引:3
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We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor. 相似文献
13.
Adenine nucleotides affect the binding of 3-phosphoglycerate to pig muscle 3-phosphoglycerate kinase
Pig muscle 3-phosphoglycerate kinase was complexed with 1-anilino-8-naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme-dye interaction did not influence the enzymic activity under the experimental conditions used. By measuring the substrate-dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (Kd less than or equal to 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20 degrees C. The dissociation constants obtained for the separate bindings of 3-phosphoglycerate, MgATP, 1,3-bisphosphoglycerate and MgADP were 0.03 +/- 0.01 mM, 0.15 +/- 0.10 mM, 0.00005 +/- 0.00001 mM and 0.15 +/- 0.10 mM respectively. binding of 3-phosphoglycerate is weakened when MgATP is also bound to the enzyme: the dissociation constant of 3-phosphoglycerate in this ternary complex (0.25 +/- 0.08 mM) is comparable to its Km value (0.38 +/- 0.10 mM). The same weakening can be observed in the non-productive ternary complexes where MgATP is replaced by MgADP (Kd = 0.20 +/- 0.10 mM) or AMP (Kd = 0.12 +/- 0.05 mM), whereas adenosine has no such effect. This indicates the importance of the negatively charged phosphate(s) of nucleotides in influencing the binding of 3-phosphoglycerate. In contrast to 3-phosphoglycerate, the binding of the substrate analogue, glycerol 3-phosphate is practically not affected by the presence of MgATP: the dissociation constant to the free enzyme (0.40 +/- 0.10 mM) is comparable to its inhibitory constant (0.70 +/- 0.20 mM). This finding and the similarity of the dissociation constant of glycerol 3-phosphate binding (0.40 +/- 0.10 mM) and the Km value of 3-phosphoglycerate (0.38 +/- 0.10 mM) suggest that, during the enzymic reaction, binding of 3-phosphoglycerate occurs probably without involvement of the carboxyl group. 相似文献
14.
The 3-phosphoglycerate kinase (EC 2.7.2.3) of yeast which contains two tryptophyl and eight tyrosyl residues per molecule, displayed an unusualy fluorescence emission spectrum with a maximum at 308 nm when excited at 280 nm. The emission peak shifted to 329 nm when excited at 295 nm. We could confirm that it was due to the efficient quenching of tryptophyl fluorescence as well as to the incomplete energy transfer from tyrosyl to tryptophyl residues. The average fluorescence quantum yield of this protein was 0.076 (excitation at 280 nm) and that of tryptophyl residues was 0.046 (excitation at 295 nm). As the pH of the solution was lowered, the fluorescence intensity of phosphoglycerate kinase at 329 nm dramatically increased between pH 5 and 4, while the position of the peak remained unchanged. When denatured in 4 M guanidine hydrochloride, the protein showed two emission peaks, one at 343 nm and the other at 303 nm. 相似文献
15.
M Vas 《European journal of biochemistry》1990,194(2):639-645
Two short analogues of 3-phosphoglycerate, -OOC-CHOH-CH2-O-PO32-, phosphonolactate, (-OOC-CHOH-CH2-PO32-) and arsonolactate (-OOC-CHOH-CH2-AsO32-) have been tested with 3-phosphoglycerate kinase. None of these served as substrate for the kinase reaction, unlike the previously studied [Orr, G. A. & Knowles, J. R. (1974) Biochem. J. 141, 721-723] analogues -OOC-CHOH-CH2-CH2-PO32- and -OOC-CHOH-CH2-CH2-AsO32-, which are isosteric with 3-phosphoglycerate. Thus, a decrease in the substrate size and the accompanying stereochemical changes cannot be tolerated by the catalytic mechanism. Instead, both analogues acted as relatively poor competitive inhibitors with respect to both 3-phosphoglycerate and MgATP. AT pH 8.5 and 20 degrees C, the inhibitory constants (Ki) of phosphonolactate and arsnolactate against both substrates are 17 +/- 5 mM and 30 +/- 7 mM, respectively. Surprisingly, however, both analogues proved to be more effective than either 3-phosphoglycerate or its isosteric analogues in protecting the enzyme against modification of its fast-reacting thiols. This comparison suggests that the shorter analogues bind differently, and that the catalytic mechanism demands a precise fitting of the -CH2-O-PO32- segment of the substrate. 相似文献
16.
Phosphoglycerate kinase levels in Hydrogenomonas facilis were reasonably constant whether cells were utilizing or synthesizing hexose during growth. Specific enzyme activities (micromoles of 3-phosphoglycerate disappearing per minute per milligram of protein) at 30 C were 0.234, 0.391, 0.300, and 0.229 in the "soluble" fraction derived from cells grown on fructose, lactate, succinate, and glutamate, respectively. The enzyme was purified 300-fold from succinate-grown cells. The final preparation, which was not homogenous but was free from glyceraldehyde-3-phosphate dehydrogenase and adenylate kinase, had a specific activity at 30 C of 90 mumoles of 3-phosphoglycerate per min per mg of protein. K(m) values for adenosine triphosphate (ATP), 3-phosphoglycerate, and Mg(++) were 0.16, 0.83, and 0.4 mm, respectively, at pH 7.4 and 30 C. Adenosine monophosphate (AMP) inhibited 23% at a ratio of AMP to ATP of 2.4, and the possible physiological implications of this inhibition are discussed. No evidence was found for an enzyme which catalyzes ATP-dependent conversion of 3-phosphoglycerate to 1,3-diphosphoglycerate, AMP, and phosphate. 相似文献
17.
3-Phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 88 units g?1 xerogel. The activity versus pH profile showed a sharper maximum at pH 6.5 in the case of the immobilized enzyme. The immobilized enzyme had a broad apparent optimum temperature range between 40 and 50°C. The apparent Km values of the immobilized 3-phosphoglycerate kinase were lower for both 3-phosphoglycerate and ATP than those of the soluble enzyme. In the case of the immobilized enzyme stabilities were enhanced. 相似文献
18.
Yeast 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phospho-transferase, EC 2.7.2.3) is inactivated by phenylglyoxal. Loss of activity correlates with the modification of two arginyl residues, both of which are protected by all of the substrates. The modification is not accompanied by any significant conformational change as determined by optical rotatory dispersion. Ultraviolet difference spectrophotometry indicates that the inactivated enzyme retains its capacity for binding the nucleotide substrates whereas the spectral perturbation characteristic of 3-phosphoglycerate binding is abolished in the modified enzyme. The data suggest that at least one of the two essential arginyl residues is located at or near the 3-phosphoglycerate binding site. A likely role of this residue could be its interaction with the negatively charged phosphate or carboxylate groups of 3-phosphoglycerate. 相似文献
19.
Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants. 总被引:3,自引:1,他引:2
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C. A. Royer C. J. Mann C. R. Matthews 《Protein science : a publication of the Protein Society》1993,2(11):1844-1852
Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389-396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683-6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741-750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
R. K. Scopes 《The Biochemical journal》1969,113(3):551-554
1. A procedure for preparing crystalline 3-phosphoglycerate kinase from rabbit or pig skeletal muscle is presented. 2. The preparation phosphorylates up to 975mumoles of 3-phosphoglycerate/min./mg. at 30 degrees and is not contaminated with myokinase. 3. The enzyme has an estimated molecular weight of 36500+/-1000, and contains three residues each of tyrosine and tryptophan. 4. The preparation is suitable for use in the enzymic procedures for determining ATP, phosphocreatine and 3-phosphoglycerate. 相似文献