Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase

Yong Kweon Cho, Terry O Matsunaga, George L. Kenyon, Byron L. Bertagnolli, Paul F. Cook

Research output: Contribution to journalArticle

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Abstract

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. The maximum rates of isotope exchange at equilibrium for the [14C]fructose 1,6-bisphosphate ⇌ fructose 6-phosphate, [32P]Pi ⇌ MgPPi, and Mg[32P]PP1 ⇌ fructose 1,6-bisphosphate exchange reactions increasing all four possible substrate-product pairs in constant ratio are identical, consistent with a rapid equilibrium mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate (F6P)/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi, in agreement with initial velocity studies [Bertagnolli, B. L., & Cook, P. F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [32P]Pi nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction.

Original languageEnglish (US)
Pages (from-to)3320-3325
Number of pages6
JournalBiochemistry
Volume27
Issue number9
StatePublished - 1988
Externally publishedYes

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pyrophosphate-fructose 6-phosphate 1-phosphotransferase
Isotopes
Kinetics
Biochemistry
Substrates
Phosphates
Fluxes
fructose-1,6-diphosphate
fructose-6-phosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cho, Y. K., Matsunaga, T. O., Kenyon, G. L., Bertagnolli, B. L., & Cook, P. F. (1988). Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase. Biochemistry, 27(9), 3320-3325.

Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase. / Cho, Yong Kweon; Matsunaga, Terry O; Kenyon, George L.; Bertagnolli, Byron L.; Cook, Paul F.

In: Biochemistry, Vol. 27, No. 9, 1988, p. 3320-3325.

Research output: Contribution to journalArticle

Cho, YK, Matsunaga, TO, Kenyon, GL, Bertagnolli, BL & Cook, PF 1988, 'Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase', Biochemistry, vol. 27, no. 9, pp. 3320-3325.
Cho, Yong Kweon ; Matsunaga, Terry O ; Kenyon, George L. ; Bertagnolli, Byron L. ; Cook, Paul F. / Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase. In: Biochemistry. 1988 ; Vol. 27, No. 9. pp. 3320-3325.
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