Structure and control of phosphofructokinase from Bacillus stearothermophilus

Evans, Philip R.; Hudson, Peter J.

doi:10.1038/279500a0

Cited by 182 publications

(150 citation statements)

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“…There, specific amino acid residues involved in catalytic and regulatory (i.e. allosteric) functions could be defined (21,22). It has been speculated that the N-terminal half of rabbit muscle Pfk retained its catalytical function, whereas in the C-terminal half a former fructose 6-phosphate binding site has evolved to a fructose 2,6-bisphosphate binding domain with allosteric function (20).…”

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A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

Heinisch

Boles

Timpel

1996

Journal of Biological Chemistry

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In this work we used in vitro mutagenesis to modify the allosteric properties of the heterooctameric yeast phosphofructokinase. Specifically, we identified two amino acids involved in the binding of the most potent allosteric activator fructose 2,6-bisphosphate. Thus, Ser 724 was replaced by an aspartate and His 859 by a serine in each of the enzyme subunits. Whereas the substitutions had no drastic effects when introduced only in one of the two types of subunits, kinetic parameters were modified when both subunits carried the mutation. Thus, the enzyme with His 859 3 Ser showed an increase in K a for binding of the activator, whereas the one with Ser 724 3 Asp failed to react to the addition of fructose 2,6-bisphosphate, at all. The enzymes still responded to other allosteric activators, such as AMP. Stabilities of the mutant subunits were not significantly altered in vivo, as judged from Western blot analysis. Phenotypically, strains expressing the mutant PFK genes showed a pronounced effect on the level of intermediary metabolites after growth on glucose. Mutants not responding to the activator at all (Ser 724 3 Asp) also displayed higher generation times on glucose medium. This could be suppressed by increasing the gene dosage of the mutant alleles. These results indicate that fructose 2,6-bisphosphate through its activation of phosphofructokinase plays an important role in regulation of the glycolytic flux.

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A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

Heinisch

Boles

Timpel

1996

Journal of Biological Chemistry

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“…Subunit interaction in EC-SOD glyceraldehyde-3-phosphate dehydrogenase (Biesecker et al, 1977); (3) loop interactions, as in triose phosphate isomerase (Banner et al, 1975); and (4) helix-helix packing, as in manganese superoxide dismutase (Borgstahl et al, 1992) and phosphofructokinase (Evans & Hudson, 1979). A structural motif able to carry out an interaction of the latter type might be a-helical coiled coil-like structures that form a-helical bundles.…”

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“…These interfaces may be viewed as a combination of four simple structural motifs: (1) extended P-sheets (sheets that form across the interface), as in Concanavalin A (Reeke et al, 1975) and prealbumin (Blake et al, 1978); (2) sheet-sheet packing, as in glyceraldehyde-3-phosphate dehydrogenase (Biesecker et al, 1977); (3) loop interactions, as in triose phosphate isomerase (Banner et al, 1975); and (4) helix-helix packing, as in manganese superoxide dismutase (Borgstahl et al, 1992) and phosphofructokinase (Evans & Hudson, 1979). A structural motif able to carry out an interaction of the latter type might be a-helical coiled coil-like structures that form a-helical bundles.…”

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Subunit interaction in extracellular superoxide dismutase: Effects of mutations in the N‐terminal domain

Stenlund¹,

Andersson²,

Tibell³

1997

Protein Science

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Human extracellular superoxide dismutase (hEC-SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC-SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X-ray structural determination have failed, the three-dimensional structure of hEC-SOD is unknown. This means that alternative strategies for structural studies are needed. The N-terminal domain of EC-SOD has already been studied using the fusion protein FusNN, comprised of the 49 N-terminal amino acids from hEC-SOD fused to human carbonic anhydrase (HCAII). The N-terminal domain in this fusion protein forms a welldefined three-dimensional structure, which probably contains a-helical elements and is responsible for the tetramerization of the protein.In this work, we have extended the studies, using site-directed mutagenesis in combination with size-exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic a-helix (formed by residues 14-32) in the N-terminal domain is essential for the subunit interaction.Keywords: carbonic anhydrase 11; extracellular superoxide dismutase; heterologous expression; site-directed mutagenesis; subunit interaction; tetramer contact area Mammalian cells produce two different types of CuZnSODs, an intracellular and an extracellular form, which are almost equally efficient as catalysts and have nearly identical kinetic parameters (Tibell et al., 1987). The two enzyme forms exhibit significant sequence similarity, but they differ in several respects. The intracellular human CuZnSOD contains 153 amino acid residues per subunit, has a subunit molecular mass of 16 kDa, and is a dimer. The mature human EC-SOD subunit, on the other hand, contains 222 amino acid residues (plus an 18-residue-long signal peptide) and a N-linked oligosaccharide, resulting in a calculated molecular mass of 26.7 kDa (24.2 kDa from the polypeptide and 2.5 kDa from the oligosaccharide) (Edlund et al., 1992). The hEC-SOD protein forms a tetramer, is secreted, and binds to proteoglycans, which are found on the surface of almost every cell in the human body. The central part of the hEC-SOD sequence is homologous with the sequence of the intracellular CuZnSODs, including those amino acid residues that are metal ligands. The 48 N-terminal Reprint requests to: LenaA.E. Tibell, Department of Biochemistry, Umei University, S-901 87 Umei, Sweden; e-mail: lenat@chem.umu.se.Abbreviarions: CuZnSOD, Cu-and Zn-containing intracellular superoxide dismutase; EC-SOD, extracellular superoxide dismutase; GuHC1, guanidine hydrochloride; HCAII, human carbonic anhydrase 11: MRW, mean residue weight: SOD, superoxide dismutase. amino acid residues of hEC-SOD, and 30 of the C-terminal, have no counterparts in intracellular CuZnSODs, and no significant homology with any other sequenced protein.The three-dimensional structure of the intrace...

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“…X-ray diffraction studies (Evans and Hudson, 1979;Shirakihara and Evans, 1988) showed that each subunit, made of two domains separated by a cleft constituting the active site, is in contact with only two of the other subunits in the tetramer. The binding site for the allosteric substrate, D-fructose 6-phosphate (Fru6P), belongs to one of these interfaces, that is therefore called the active (A) interface, whereas the effector binding site belongs to the other, so-called regulatory (R) interface.…”

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Reversible high hydrostatic pressure inactivation of phosphofructokinase from Escherichia coli

Deville‐Bonne

Else

1991

European Journal of Biochemistry

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Tetrameric Escherichia coli phosphofructokinase dissociates reversibly on incubation under hydrostatic pressures of 80 MPa and above, yielding inactive dimers and monomers. The transition is dependent upon enzyme concentration and presence of ligands. The substrate, o-fructose 6-phosphate, which bridges the intersubunit interface at the active site, produces a massive stabilization to pressure, whereas ATP, which binds to only one subunit, induces only a mild stabilization. Both the positive allosteric regulator, GDP, and the negative allosteric regulator, phosphoenolpyruvate, whose binding sites lie at the other subunit interface, produce an intermediate effect. Of these ligands, only ATP increases the rate of reactivation after depressurization.Phosphofructokinase 1 (PFK) catalyses the phosphorylation reaction that controls glycolysis:Fructose 6-phosphate + ATP + Fructose 1,6-bisphosphate + ADP.The enzyme is regulated by allosteric effectors: the activators (ADP, GDP) and the inhibitor phosphoenolpyruvate (PPrv) bind to the same effector site in an exclusive way.Escherichia coli PFK is a tetramer of identical polypeptide chains of M , 34817 (Hellinga and Evans, 1985) whose sequence is known. X-ray diffraction studies (Evans and Hudson, 1979;Shirakihara and Evans, 1988) showed that each subunit, made of two domains separated by a cleft constituting the active site, is in contact with only two of the other subunits in the tetramer. The binding site for the allosteric substrate, D-fructose 6-phosphate (Fru6P), belongs to one of these interfaces, that is therefore called the active (A) interface, whereas the effector binding site belongs to the other, so-called regulatory (R) interface.

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Structure and control of phosphofructokinase from Bacillus stearothermophilus

Cited by 182 publications

References 29 publications

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

Subunit interaction in extracellular superoxide dismutase: Effects of mutations in the N‐terminal domain

Reversible high hydrostatic pressure inactivation of phosphofructokinase from Escherichia coli

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