Phosphofructokinase: Complete Amino-Acid Sequence of the Enzyme from Bacillus stearothermophilus

Kolb, E.D.; Hudson, Peter J.; Harris, J. Ieuan

doi:10.1111/j.1432-1033.1980.tb04754.x

Cited by 38 publications

(15 citation statements)

References 17 publications

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“…mHElOll is now being used for making oligonucleotide-directed point mutations in the pfkA gene to construct phosphofructokinase-1 mutant enzymes. The fragments carrying the mutations can then be recloned into pUC9 for overproduction of the mutant enzymes, thereby greatly Three other phosphofructokinase protein sequence have been determined: the sequence of the E. coli phosphofructokinase-2 [12], that of the enzyme isolated from the thermophile B. stearothermophilus [36] and most of the sequence of the rabbit muscle enzyme [45]. Comparison of the E. coli phosphofructokinase-2 enzyme sequence to any of the others showed no homologous features, indicating that although the reaction catalysed is the same, this enzyme has a different evolutionary origin.…”

Section: Comparison Of the Known Phosphofructokinase Amino Acid Sequementioning

confidence: 99%

“…4). The pfkA reading frame was identified by the sequence homology of its predicted translation product to the known amino acid sequence of the Bacillus stearothermophilus phosphofructokinase [36]. The second open reading frame was identified as a periplasmic sulphatebinding protein by its homology to the amino acid sequence of such a protein isolated from Salmonella typhimurium [37] (see Fig.…”

Section: Location and Identification Of Coding Regionsmentioning

confidence: 99%

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Nucleotide sequence and high‐level expression of the major Escherichia coli phosphofructokinase

Hellinga

Evans

1985

European Journal of Biochemistry

135

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The gene for the major phosphofructokinase enzyme in Escherichia coli, pfk A, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA – tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate‐binding protein, sbp, and for a membrane‐bound enzyme, CDP‐diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA‐sbp‐cdh‐tpi. The pfkA gene has been subcloned into a high‐copy‐number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 × 103‐nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40–50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using ‘reverse genetics’.

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Section: Comparison Of the Known Phosphofructokinase Amino Acid Sequementioning

confidence: 99%

Section: Location and Identification Of Coding Regionsmentioning

confidence: 99%

Nucleotide sequence and high‐level expression of the major Escherichia coli phosphofructokinase

Hellinga

Evans

1985

European Journal of Biochemistry

135

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“…Indeed, the PFKs from Lactobacillus bulgaricus and Spiroplasma citri, which do not undergo an R --T transition (27,29) have, respectively, Asp and Ile at position 241 (22,23) while Glu-241 is present in the PFKs from E. coli (20), B. stearothermophilus (19), and Thermus thermophilus (22), which respond to PEP binding by an R --T transition (3,12,30). It is more difficult to predict the effect of a mutation of Arg-72 since this residue is engaged in an ionic bond in both the R and T states, albeit with different partners.…”

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confidence: 99%

The cooperativity and allosteric inhibition of Escherichia coli phosphofructokinase depend on the interaction between threonine-125 and ATP.

Auzat

Bras²,

Garel³

1994

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

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During the reaction catalyzed by the phosphofructokinase (EC 2.7.1.11) from Escherichia coli, the phosphoryl group transferred from ATP interacts with [Shir a, Y. & Evans, P. R. (1988) J. Mol. Biol. 204,. The replacement of Thr-125 by serine changes the saturation by fructose 6-phosphate from cooperative to hyperbolic and abolishes the allosteric inhibition by phosphoenolpyruvate. The same changes, a saturation by fructose 6-phosphate that is no longer cooperative and an activity that is no longer inhibited by phosphoenolpyruvate, are observed with wild-type phosphofructokinase when adenosine 5'-[r thio]triphosphate is used instead of ATP as the phosphoryl donor. These two perturbations of the ATP-Thr-125 interaction lead to the suppression of both the allosteric inhibition by phosphoenolpyruvate and the cooperativity of fructose-6-phosphate saturation, as if replacing the neutral oxygen of ATP by sulfur or removing the methyl group of Thr-125 had "locked" phosphofructokinase in its active conformation. The geometry ofthis ATP-Thr-125 interaction and/or the presence of the methyl group on the P-carbon of Thr-125 are crucial for the regulatory properties of phosphofructokinase. This interaction could be a hydrogen bond between the neutral oxygen of the rphosphate of ATP and-the hydroxyl group of Thr-125.The regulation of many biological processes involves allosteric interactions between distinct sites of the same protein, such that ligand binding at one site modifies the functional properties at a distant site. Cooperativity is observed when positive interactions take place between identical sites (1). One ofthe "classic" cooperative enzymes is Escherichia coli phosphofructokinase (PFK; ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) (2, 3), which shows a markedly sigmoidal saturation by its substrate fructose 6-phosphate (Fru-6P), with a Hill cooperativity coefficient close to nH = 4 for four Fru-6P sites (3). This enzyme is also sensitive to allosteric effectors, which bind to a regulatory site remote from the active site; PFK is activated by ADP (or GDP) and inhibited by phosphoenolpyruvate (PEP) (3).The steady-state kinetics of E. coli PFK have been interpreted according to the concerted allosteric mechanism (4), in which the protein is in equilibrium between two states, an active R state, which binds Fru-6P and the activator ADP or GDP, and an inactive T state, which binds the inhibitor PEP (3). X-ray crystallography shows that PFK exists in two different conformations, R with activator and substrate (or products) bound (5) and T with inhibitor bound (6), which provides a structural explanation for the allosteric inhibition by PEP (6-8). However, in contrast with hemoglobin for which the differences between the oxy and deoxy states could largely explain the cooperativity of oxygen binding (8,

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“…The ORF III protein, which remains unidentified, has been named protein X. The protein encoded by ORF V had 45.8% homology with the sequence of PFK from Bacillus stearothermophilus (Bs-PFK) (16) (Fig. 6C).…”

Section: Resultsmentioning

confidence: 99%

Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein

Chevalier¹,

Saillard²,

Bové³

1990

J Bacteriol

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The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf-X-spiralin gene-pfl-pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction.Spiroplasmas are mollicutes (mycoplasmas) (class Mollicutes) with motility and helical morphology (33). Analysis of the Spiroplasma genome by classical genetic techniques has been difficult. The dependence of these organisms on complex growth media and their poorly defined metabolic pathways explain the paucity of auxotrophic mutants and hence of genetic markers in these procaryotes. In the absence of such markers, characterization of cloned DNA fragments provides the means for analyzing the structural organization of Spiroplasma genes.The gene for spiralin seemed to be of particular interest. Spiralin (35) is the major membrane protein of Spiroplasma citri, an important plant pathogen (28). It is apparently a transmembrane amphiphilic protein (34), and it is acylated (37). Acylation seems to be a characteristic of several membrane proteins of mollicutes. The role of spiralin is not known. It must, however, be a key protein of the spiroplasmal membrane, since spiralinlike proteins occur in Spiroplasma species other than S. citri (37) and probably in all spiroplasmas. Elucidation of the amino acid sequence of spiralin as deduced from the nucleotide sequence of its gene would undoubtly contribute to a better understanding of its structure and function, as well as the organization of its gene. Sequence determination of the spiralin gene was made possible by the fact that we have previously cloned the gene.

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Phosphofructokinase: Complete Amino-Acid Sequence of the Enzyme from Bacillus stearothermophilus

Cited by 38 publications

References 17 publications

Nucleotide sequence and high‐level expression of the major Escherichia coli phosphofructokinase

Nucleotide sequence and high‐level expression of the major Escherichia coli phosphofructokinase

The cooperativity and allosteric inhibition of Escherichia coli phosphofructokinase depend on the interaction between threonine-125 and ATP.

Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein

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