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

Abstract

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.

Different Physiological Roles of ATP- and PPi-Dependent Phosphofructokinase Isoenzymes in the Methylotrophic ActinomyceteAmycolatopsis methanolica

“…The latter enzyme is not sensitive to PEP and only slightly inhibited by ATP and F-1,6-P 2 . ADP affects PFK2 activity, with up to 50% inhibition at low F-6-P and ATP concentrations (17). It is unclear whether these two E. coli PFKs have distinct physiological roles (11).…”

Are the Aerobic and Anaerobic Phosphofructokinases of Escherichia coli Different?

“…(a) Phosphofructokinase-I is easy to desensitize [lo]. (b) It needs NH: or K + for maximal activity [lo], a requirement not always recognized [3,7,8]. One of these ions in sufficient amount may be supplied by the other reagents in an uncontrolled way.…”

“…One of these ions in sufficient amount may be supplied by the other reagents in an uncontrolled way. (c) Since ADP activates, the assay (at least at low fructose-6-P concentration) needs an ATP-generating system [7]; this was not used [1,3]. With impure enzymes it is even more difficult to assess the effect of adenine nucleotides [19].…”

“…Earlier, with publication of [l], we purified phosphofructokinase from aerobic and anaerobic cultures, but found that both in kinetics and course of purification the predominant activity from each culture was typical phosphofructokinase-I (unpublished). With the appearance of the detailed report describing the different aerobic and anaerobic enzymes [3], we have reinvestigated the matter, this time using chemostat cultures (as in [l]; our earlier work, as well as [3], used batch cultures). We find again that with respect to kinetics and subunit size the pure enzymes from aerobic and anaerobic cultures do not differ: they are both phosphofructokinase-1.…”

Evidence for the Existence of Two Interconvertible Forms of the Phosphofructokinase Dimer from Escherichia coli K‐12

“…Both enzyme proteins were separated and partially purified [2,3]. As a result of continuous culture experiments it was suggested that the cell induces a separate dimeric form of phosphofructokinase depending upon the prevailing oxygen conditions [ 11.…”