Manganese substrate complexes of phosphofructokinase studied by pulsed magnetic resonance

G. Larry Cottam, Kosaku Uyeda

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

The formation of binary, ternary, and quaternary complexes between phosphofructokinase, manganese, and substrates has been demonstrated by use of pulsed nuclear magnetic resonance techniques. A Scatchard plot of the interaction of manganese with phosphofructokinase as determined by electron paramagnetic resonance shows two types of manganese binding sites. Phosphofructokinase seems to contain one or two of the metal binding sites with Kd = 20 μm and ε{lunate}b ≦ 4, and perhaps, as many as 14 binding sites with Kd ~ 0.8 mm and ε{lunate}b ≦ 12 ± 2 per enzyme. Addition of ATP or ADP results in a further enhancement of the relaxation rate indicating ternary complex formation. The concentration of ATP and ADP which results in half maximal change of enhancement is 30-100 μm and 80 μm, respectively. No change in the water proton relaxation rate was detected upon addition of fructose-6-P or fructose-1,6-bisphosphate. A quaternary complex was detected by proton relaxation measurements upon addition of fructose-6-P to a reaction mixture containing β, γ-methylene ATP, manganese, and enzyme with 50 μm fructose-6-P required to obtain the half maximal observed effect. This evidence for a quaternary complex is consistent with a sequential reaction mechanism for phosphofructokinase.

Original languageEnglish (US)
Pages (from-to)683-690
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume154
Issue number2
DOIs
StatePublished - Feb 1973

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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