Sequence and structure of yeast phosphoglycerate kinase.

Watson, Herman C.; Walker, ND; Shaw, Pangchui; Bryant, T. N.; Wendell, P.L.; Fothergill, Linda A.; Perkins, R E; Conroy, Stephen C.; Dobson, Melanie J.; Tuite, Mick F.

doi:10.1002/j.1460-2075.1982.tb01366.x

Cited by 363 publications

(251 citation statements)

References 25 publications

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“…The GAPDH crystal structure is tetrameric (46), and has been shown to exist as the tetramer GAPDH 4 under crowded conditions (47). PGK is known to exist as a monomer in the cytoplasm and has been extensively studied by us and others (31,48,49). These proteins have been shown to interact in vitro by using gel and affinity chromatography (50,51).…”

Section: Heterooligomerization Of Fps Can Be Detected By Cell-volumementioning

confidence: 99%

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

Sukenik

Ren

Gruebele

2017

Proc. Natl. Acad. Sci. U.S.A.

104

123

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Weakly bound protein complexes play a crucial role in metabolic, regulatory, and signaling pathways, due in part to the high tunability of their bound and unbound populations. This tunability makes weak binding (micromolar to millimolar dissociation constants) difficult to quantify under biologically relevant conditions. Here, we use rapid perturbation of cell volume to modulate the concentration of weakly bound protein complexes, allowing us to detect their dissociation constant and stoichiometry directly inside the cell. We control cell volume by modulating media osmotic pressure and observe the resulting complex association and dissociation by FRET microscopy. We quantitatively examine the interaction between GAPDH and PGK, two sequential enzymes in the glycolysis catalytic cycle. GAPDH and PGK have been shown to interact weakly, but the interaction has not been quantified in vivo. A quantitative model fits our experimental results with log K d = −9.7 ± 0.3 and a 2:1 prevalent stoichiometry of the GAPDH:PGK complex. Cellular volume perturbation is a widely applicable tool to detect transient protein interactions and other biomolecular interactions in situ. Our results also suggest that cells could use volume change (e.g., as occurs upon entry to mitosis) to regulate function by altering biomolecular complex concentrations.quinary interactions | protein-protein interactions | cell volume | FRET | live-cell microscopy F rom forming active enzymatic complexes to facilitating signal transduction in regulatory networks, protein interactions are pivotal to cell function. Strong interactions, with dissociation constants (K d ) of nanomolar and lower (1, 2), can be measured accurately both in vitro and in vivo (3, 4). These interactions are ideal in cases where the complex should form with high fidelity and persist. The downside of strong binding is that its regulation in the cellular environment is limited: Once a complex is formed, it seldom dissociates.Another type of protein complexes relies on weak, transient interactions, collectively termed quinary interactions (5-7). Such interactions, with a K d in the micromolar to millimolar range, are emerging as important components of the cell's signaling, regulatory, and stress adaptation mechanisms (6,8,9). Their transient nature is key to their function: Unlike tightly bound complexes, quinary interactions are highly sensitive to variations in their environment and respond rapidly to changes in temperature, pressure, pH, or the local concentration of surrounding molecules. The sensitivity of quinary interactions makes them important in fine-tuning cellular processes. For example, it has been proposed that sequential metabolic enzymes could associate to improve substrate transfer between catalytic enzymes (10), that weak protein association can mediate cytokine release (11), or that phase-separated protein droplets held together by quinary interactions could serve for cellular storage or stress response (12, 13).Despite growing interest, quantification of quinary inte...

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Section: Heterooligomerization Of Fps Can Be Detected By Cell-volumementioning

confidence: 99%

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

Sukenik

Ren

Gruebele

2017

Proc. Natl. Acad. Sci. U.S.A.

104

123

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“…These highly conserved regions in GAPDH contain those amino acids postulated to be implicated in the NAD-binding Pap fold (residues 4 to 20, and especially Arg-12, Ile-13, Asp-36, Arg-82, Ser-121, Asn-315, and Tyr-319 in the C. glutamicum sequence) or in the catalytic mechanism (Ser-152, Cys-153, Thr-154, His-180) (8,53). In PGK, the conserved regions contain those sites which in yeast and horse muscle enzymes have been shown to be important for structural or catalytic properties (4,26,41,58) (1,5,43).…”

Section: E--tsk-d----ea--e-rn Rfaqelaala Adngafvsdg Fgvvhraqts -Vydiamentioning

confidence: 99%

Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase

Eikmanns

1992

J Bacteriol

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To investigate a possible chromosomal clustering of glycolytic enzyme genes in Corynebacterium glutamicum, a 6.4-kb DNA 27,198). The amino acid sequences of the three enzymes show up to 62% (GAPDH), 48% (PGK), and 44% (TPI) identity in comparison with respective enzymes from other organisms. The gap, pgk, tpi, and ppc genes were cloned into the C. glutamicum-Escherichia coli shuttle vector pEKO and introduced into C. glutamicum. Relative to the wild type, the recombinant strains showed up to 20-fold-higher specific activities of the respective enzymes. On the basis of codon usage analysis of gap, pgk, tpi, and previously sequenced genes from C. glutamicum, a codon preference profile for this organism which differs significantly from those of E. coli and Bacillus subtilis is presented.

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“…Being intracellular, it contains no disulphide bonds which could also cause difficulties in thermal-denaturation studies. PGK has been purified from a wide variety of sources [1][2][3], and its conserved three-dimensional structure is known [4][5][6][7][8]. Detailed kinetic and mechanistic investigations have been conducted with the yeast enzyme [9][10][11][12][13][14][15], and most aspects of its kinetic behaviour are understood.…”

Section: Introductionmentioning

confidence: 99%

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

Thomas

Scopes

1998

Biochemical Journal

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The effects of temperature on the kinetic parameters kcat and Km, for three isolates of the highly conserved monomeric enzyme 3-phosphoglycerate kinase (PGK), were investigated in detail using a rapid automated kinetics apparatus. PGK was purified from the thermophilic bacterium Thermoanaerobacter sp. Rt8.G4 (optimum growth temperature 68 °C), the mesophile Zymomonas mobilis (optimum growth temperature 32 °C) and a second, unidentified, soil mesophile designated unid A (optimum growth temperature 27 °C). The kinetic behaviour with temperature of each PGK preparation was distinct, despite the conserved nature of the enzyme. The kcat values increased with temperature, but not as rapidly exponentially, as might be expected from the Arrhenius equation. Maximum kcat values were at much higher temperatures than the optimum growth temperatures for the mesophiles, but for the thermophile the temperature of maximum kcat was close to its optimum growth temperature. Km values were in general nearly constant through the lower temperature ranges, but increased substantially as the optimum temperature (highest kcat) was passed. Thermal irreversible denaturation of the PGK proteins was also investigated by measuring loss of activity over time. In a dilute buffer, Arrhenius plots for denaturation were linear, and the calculated apparent energy of activation (Eact) for denaturation for the thermophilic PGK was 600 kJ·mol-1, whereas for the mesophilic enzymes the values were 200-250 kJ·mol-1. In the presence of substrates, a considerable stabilization occurred, and in the case of the Z. mobilis enzyme, the apparent Eact was increased to 480 kJ·mol-1. A theoretical explanation for these observations is presented. Comparing the kinetics data with irreversible denaturation rates determined at relevant temperatures, it was clear that kcat values reached a maximum, and then decreased with higher temperature before irreversible denaturation had any significant influence.

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Sequence and structure of yeast phosphoglycerate kinase.

Cited by 363 publications

References 25 publications

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

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