Yeast phosphofructokinase: Effector-controlled conversion of an ATP-sensitive to an ATP-desensitized form

Afting, E G; Ruppert, D.; Hagmaier, V.; Holzer, Helmut

doi:10.1016/0003-9861(71)90243-8

Cited by 28 publications

(7 citation statements)

References 12 publications

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“…The results reported in this paper confirm the existence of two different types of phosphofructokinases, an ATP-sensitive and an ATP-insensitive enzyme [1,4,14,19,20]. The formation of these two types depends upon the oxygen tension in the cultivation medium.…”

Section: Discussionsupporting

confidence: 77%

“…In recent years phosphofructokinase studies with yeast have led to the observation of ATP-desensitization [14,19,20], with the suggestion that this desensitization is catalyzed by a specific enzyme [27], although recent observations have shown [19] that such an enzyme is not necessary for desensitization. This conversion from the sensitive to the ATPdesensitized enzyme occurs only in the presence of a heat-labile fraction from yeast, and the non-inhibition by ATP can only be retained in the presence of 10-20 mM NaF [28].…”

Section: Discussionmentioning

confidence: 99%

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ATP‐Sensitive and ATP‐Insensitive Phosphofructokinase in Escherichia coli K‐12

Doelle

1975

European Journal of Biochemistry

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The purification and kinetic characteristics of two phosphofructokinases are described. Aerobic cultures of Escherichia coli exhibit two types of phosphofructokinase. Both types are dimers of mol. wt 150000 (subunit mol. wt 73000), whereas the anaerobic culture of E. coli revealed only one type, which is a tetramer of mol. wt 350000 (subunit mol. wt 90000). Type 1 of the aerobic enzyme, representing approximately 70 % of the total enzyme activity, is ATP-insensitive, whereas type I1 and the anaerobic enzyme are ATP-sensitive.The addition of AMP stimulates the tetramer, relieving ATP inhibition, and also the type I1 dimer, which is, however, inhibited at concentrations higher than 0.5 mM AMP. No effect was observed on the type I dimer of the aerobic preparation. ADP stimulates the tetramer and inhibits type I more strongly than type I1 of the aerobic dimer.The kinetic characteristics together with the effect of metabolites on these phosphofructokinase types are described and discussed in the light of their importance for the regulatory mechanism of the Pasteur effect.Investigations into the effect of oxygen an aerobic and anaerobic glucose utilization of Escherichia coli K-12 [l, 21 revealed the existence of an ATP-sensitive and ATP-insensitive phosphofructokinase depending upon the oxygen tension in the culture medium. Both forms of phosphofructokinase reacted differently in regard to nucleotide modulators, but were not effected by increased glucose concentration [3]. The existence of two phosphofructokinase activities was also observed in an unlinked suppressor mutation RfkBI [4]. As phosphofructokinase is regarded as the key enzyme for the regulation of the Pasteur effect, the observation of two forms would give further evidence in support of the hypothesis [5,6] that the rate of the reaction depends not only on the activity of the enzyme as controlled by ATP, ADP and fructose 6-phosphate concentrations, but also on the concentration and the type or form of phosphofructokinase protein in the cell. This paper gives further evidence for the existence of two forms of phosphofructokinase in Escherichia coli K-I 2, their partial purification, kinetic characteristics and significance in the mechanism of the Pasteur effect.

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Section: Discussionsupporting

confidence: 77%

Section: Discussionmentioning

confidence: 99%

ATP‐Sensitive and ATP‐Insensitive Phosphofructokinase in Escherichia coli K‐12

Doelle

1975

European Journal of Biochemistry

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“…The net decline in the rate of reaction a t the end of 2 min would be only 25 to 30°/, if it were to be entirely due to the depletion of substrates. When this enzyme was incubated for 25 min a t (27-29 "C) in the presence of ADP, fructose 6-phosphate, sodium fluoride and magnesium chloride which are part of the mixture used to desentize yeast phosphofructokinase to ATP inhibition [19] and assayed a t 0.1 mM fructose 6-phosphate and 0.083 mM ATP, the rate declined only by 2501, by the end of 2 min during which time 33O/, of initial concentration of fructose 6-phosphate and 40°/, of ATP were used.…”

Section: Effect Of Fructose 6-phosphate and Atp On The Rate Of Transimentioning

confidence: 99%

Interconvertible Forms of Phosphofructokinase of Rabbit Liver

Ramaiah

Tejwani

1973

European Journal of Biochemistry

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When the phosphofructokinase reaction was started with the addition of stock rabbit liver phosphofructokinase its activity declined rapidly, a t low concentrations of fructose 6-phosphate or a t high concentrations of ATP. The rate of this decline is independent of enzyme concentration and it could be prevented by the presence of positive effectors of the enzyme or by high concentration of fructose 6-phosphate.When the reaction rate was measured in the first few seconds after addition of stock enzyme, the activity of the enzyme and its [S],., value (concentration of fructose 6-phosphate for half maximal velocity) were not affected significantly by the presence of positive effectors of the enzyme. The stock enzyme, which was routinely stored in the presence of 0.7M (NH,),SO,, i0mM K,HPO,, 1OmM ATP, 50mM Tris-HC1 pH 7.5 and 0.33mM EDTA, was transformed to a less active form of enzyme, which had a very high [S],., value and low intrinsic activity on dilution or when freed of effectors by passing the enzyme solution through a Sephadex G-25 column. This form can be converted back to the active form by the positive effectors of the enzyme. The rate of this conversion of less active form to the active form of enzymes is again independent of enzyme concentration.The rate-limiting step in the interconversions of active form to less active form of enzyme or vice versa is a first-order process and there appears to be no aggregation or disaggregation of the enzyme during these interconversions.A variety of [S],., values and maximal velocities were observed for the enzyme depending on the preincubation, prior dilution andsemoval of the effectors of enzyme suggesting the existence of a t least two conformational states of enzyme in varying proportions differing widely in their It was shown earlier that partially purified rabbit liver phophofructokinase stored in the presence of NH,+, Kf, SO,,-, Pi and ATP exists in a form with high affinity for fructose 6-phosphate and on dilution is transformed to a form with affinity for fructose 6-phosphate a t pH 7.5 [1,2]. It was suggested that these observations may be interpreted on the basis of slow conformational change( s) or aggregationdisaggregation of the enzyme or both, as a result of changes in the concentration of enzyme and effectors.Enzyme. Phosphofructokinase or ATP : D-fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.11).Definition.[S],.,, the concentration of fructose 6-phosphate required for half maximal velocity under the given conditions of assay.I n this article it is shown that the presence of effectors in the stock enzyme solution keeps the enzyme in the active state with high intrinsic activity and affinity for fructose 6-phosphate, and when these effectors are removed either by dilution or by passing the enzyme through Sephadex G-25, the enzyme is transformed slowly to a form with very low affinity for fructose 6-phosphate and relatively low intrinsic activity. The less active form of enzyme could be converted back to the active form by the positive effectors o...

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“…Phosphofructokinase activity was assayed as described by Afting et alJ 14 The molecular weight of phosphofructokinase was determined by the method of Martin and Amesl 18 ! cerevisiae, strain X 2180 B, was grown in a complete medium containing 1 % Difco yeast extract, 2% Difco bactopeptone and 2% glucose and harvested during the late logarithmic growth phase.…”

Section: Methodsmentioning

confidence: 99%