The phosphofructokinase genes of yeast evolved from two duplication events

Heillisch, Jürgen; Ritzel, R. Gary; Borstel, R. C. von; Aguilera, Andrés; Rodicio, Rosaura; Zimmermann, Friedrich K.

doi:10.1016/0378-1119(89)90233-3

Cited by 89 publications

(64 citation statements)

References 45 publications

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“…A globular protein having an elution time of 8.55 min would have an apparent molecular mass of about 300 m a , characteristic of the tetrameric 12 S form of yeast phosphofructokinase (see Table 1). steat-othermophilus and human and rabbit muscle according to Heinisch et al (1989).…”

Section: Exposure To Chymotrypsin and To Trypsinmentioning

confidence: 99%

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Limited proteolysis of yeast phosphofructokinase

Kopperschläger

Bär

Stellwagen

1993

European Journal of Biochemistry

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Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerevisiae) was subjected to proteol ysis by thermolysin, endoproteinase lys-C, trypsin and chymotrypsin under defined solvent conditions. In the absence of substrates and allosteric effectors, the catalytic activity of phosphofructokinase rapidly disappeared in the presence of each proteolytic enzyme. The presence of a saturating concentration of ATP protected phosphofructokinase activity from proteolytic inactivation while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate provided transient activation during proteolysis.Changes in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromatography while changes in the primary sequence of the individual a and p polypeptide chains were estimated by polyacrylamide-gel electrophoresis in sodium dodecylsulfate. The site(s) of proteolytic cleavage were identified by N-terminal sequence analysis of resolved electrophoretic components.The presence of ATP protects phosphofructokinase from thermolysin proteolysis, while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate restricts proteolysis to one site in each polypeptide chain involving the peptide bonds preceding Leu199 in the a chain and Leu192 in the p chain. The truncated phosphofructokinase retains its octameric structure. The presence of ATP largely restricts endoproteinase lys-C proteolysis to a single site in the n chain involving the peptide bond preceding Va1914. This cleavage results in the dissociation of the octameric form of phosphofructokinase into two tetramers. The presence of ATP restricts both trypsin and chymotrypsin proteolysis to the N-terminal and C-terminal regions described above, resulting in the preferential stabilization of the tetrameric form of phosphofructokinase.It would appear that the first 200 and last 80 residues which are unique to the sequence of the yeast phosphosphofructokinase are not directly involved in catalysis or its allosteric regulation.However, the last 80 residues of the a polypeptide chain do appear to stabilize an octameric structure which is unique to yeast phosphofructokinase.Yeast phosphofructokinase 1 contains two kinds of polypeptide chains, an a chain containing 987 residues in a known sequence and encoded by the PFKl gene and a chain containing 959 residues, also in a known sequence encoded by the PFK2 gene (Heinisch et a]., 1989). The native enzyme is a globular protein containing four a chains and four p chains. Solution measurements indicate the native octameric enzyme has a sedimentation coefficient of 21 S and a molecular mass of about 800 kDa (Kopperschlager et al., 1977;Chaffotte et al., 1984). The octameric structure of the native enzyme is stable at a protein concentration of 1 mg/ ml at ambient temperature and neutral pH in the presence or Correspondence to

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Section: Exposure To Chymotrypsin and To Trypsinmentioning

confidence: 99%

“…Yeast phosphofructokinase 1 contains two kinds of polypeptide chains, an a chain containing 987 residues in a known sequence and encoded by the PFKl gene and a chain containing 959 residues, also in a known sequence encoded by the PFK2 gene (Heinisch et a]., 1989). The native enzyme is a globular protein containing four a chains and four p chains.…”

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confidence: 99%

Limited proteolysis of yeast phosphofructokinase

Kopperschläger

Bär

Stellwagen

1993

European Journal of Biochemistry

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“…The latter have been cloned, sequenced, and deleted from the haploid yeast genome (12,13). Whereas in most organisms homotetrameric enzymes are usually active, mutations in one of the yeast genes lead to a loss of in vitro detectable Pfk activity (14 -16).…”

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confidence: 99%

“…Comparison of the primary sequence of Bacillus stearothermophilus Pfk with the one from rabbit muscle led to the notion that the latter evolved through a gene duplication event from a prokaryotic ancestor (20). This was also postulated for each of the yeast Pfk subunits, where apparently a second gene duplication followed, locating the two genes on different chromosomes (13).…”

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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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“…RESULTS AND DISCUSSION 3.1. Sequencing of the PFKI-cud PFK2-promoters Published work [3] contains little information on the 5'-non-ceding regions of the two PFK genes. The PFKl sequence was determined up to -260 bp relative to the ,4TG translation start codon and the PFK2 sequence up to -420 bp.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional control of yeast phosphofructokinase gene expression

1991

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We here provide the complete nucleotidc sequences of the 5'-non-coding regions of the yeast phosphofructokinase genes. PFKI and PFK2. /rcZ fusions of the PFKl and PFKJ promoters were constructed and a deletion analysis was performed. In contrast to other glycolytic gene promoters. no strong regulatory elements could be found. However. we detected moderate UAS and URS functicns. In general. the effects on expression upon deletion of these regions were more pronounced on media containing ethanol than on those containing glucose as carbon sources. Overespression of either one of the PFK genes led to a decreased enzymatic activity in a wild-type background but did not affect transcription from the promoters.

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The phosphofructokinase genes of yeast evolved from two duplication events

Cited by 89 publications

References 45 publications

Limited proteolysis of yeast phosphofructokinase

Limited proteolysis of yeast phosphofructokinase

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

Transcriptional control of yeast phosphofructokinase gene expression

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