Binding of Metabolites by Phosphofructokinase<sup>*</sup>

Kemp, Robert G.; Krebs, Edwin G.

doi:10.1021/bi00854a009

Cited by 169 publications

(72 citation statements)

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“…The present kinetic studies and equilibrium binding studies [16] indicate that the protomer of 90000 molecular weight has one binding site for fructose 6-phosphate, the second substrate, and hence the protomer is likely to have only one catalytic binding site for Mn-ATP. Within the limits of error of the NMR study, we have detected two equivalent sites for Mn-ATP per 90000 molecular weight.…”

Section: Discussionmentioning

confidence: 60%

“…The Hill coefficients approach4 as the pH is decreased and are thus compatible with a system involving a high degree of cooperativity between four Fru-6-P sites on the fully active oligomer of molecular weight 360000. This is the number of binding sites found by Kemp in equilibrium binding studies [16]. The extreme sensitivity of the regulation as a function of pH has been stressed before [3,4]; in the data shown there is more than a 100-fold change in activity a t low Fru-6-P concentrations between pH 6.5 and 7.0.…”

Section: Kinetics Of the Manganese-activated Enzymementioning

confidence: 76%

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Magnetic Resonance Studies on Manganese‐Activated Phosphofructokinase

Jones

Dwek

Walker

1972

European Journal of Biochemistry

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Rabbit skeletal-muscle phosphofructokinase exhibits similar kinetic behaviour and specific activity when manganese is used as the essential divalent cation in place of magnesium. At pH 7.5, the kinetics are Michaelis-Menten with respect to fructose 6-phosphate, ATP and manganous ion concentrations ; the reaction, as a function of metal ion concentration, is inhibited competitively by excess free ATP and by calcium ions. Between pH 7.0 and 6.5, in the presence of high ATP concentrations, there is a sharp transition to sigmoid kinetics as indicated by an increase of the Hill interaction coefficient from 1 to 4.1. Preincubation of the enzyme with ligands has a marked effect on activity, particularly free divalent metals which cause dissociation and an almost total loss of activity.Studies on the rates of water-proton longitudinal relaxation a t pH 7.5 and 35 MHz indicate that, in the absence of nucleotides, the phosphofructokinase-manganese system shows low relaxation enhancements (&b m 2). On titrating with ATP a t fixed enzyme and manganese ion concentrations, the enhancement rises sharply, then falls again at very high ATP levels. An analysis of enhancement data a t fixed ATP concentrations as a function of enzyme and metal ion concentrations, using a combination of graphical and computational methods, indicates that the protomer of 90000 molecular weight has 2 Mn-ATP sites with dissociation constant 240 pM and an enhancement for the ternary complex Et, of 18. Free ATP binds competitively with a dissociation constant of 3 mM. Line-broadening studies on the H-2 and H-8 resonances of the adenine ring in the high resolution spectrum of ATP reveal that the proton-manganese distances in Mn-ATP are almost unchanged in forming the ternary complex with enzyme. The kinetic and magnetic resonance data indicate that Mn-ATP is the true substrate, which probably binds to the enzyme via a nucleotide bridge.The kinetic activity of phosphofructokinase from a great variety of sources has been studied with particular regard to its role in the control of glycolysis. Studies on the enzyme from mammalian skeletal muscle have been mainly involved with its characterisation [l], subunit structure [2], the regulatory properties [3], the effects of chemical modification [4,5] and the reaction mechanism [6]. All the studies on activity have used magnesium as the essential divalent cation. The metal ion is thought to bind to the enzyme in the form of a complex with ATP which is the true substrate.A preliminary observation made in this laboratory [7] showed that the enzyme was highly active when manganese replaces magnesium as the essential diAbbreviations. NMR, nuclear magnetic resonance; ESR, electron spin resonance; Fru-6-P, fructose 6-phosphate.Enzymes. Phosphofructokinase (EC 2.7.1.11) ; creatine kinase (EC 2.7.3.2) ; pyruvate kinase(EC 2.7.2.40) ; aldolase orketose-l-phosphate aldehyde-lyase (EC 4.1.2.7) ; triosephosphate isomerase (EC 5.3.1.1); glycerol-l-phosphate dehydrogenase (EC 1.1.1.8).valent cation. This opened up the possibil...

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Section: Discussionmentioning

confidence: 60%

Section: Kinetics Of the Manganese-activated Enzymementioning

confidence: 76%

Magnetic Resonance Studies on Manganese‐Activated Phosphofructokinase

Jones

Dwek

Walker

1972

European Journal of Biochemistry

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“…Citrate inhibits PFK by binding directly to PFK, which decreases the enzyme's affinity for its substrate, F6P, and its activator, ADP. Citrate also increases the enzyme's affinity for ATP at the substrate site and, more important, at the inhibitory site (Kemp and Krebs, 1967).…”

Section: Introductionmentioning

confidence: 99%

“…This enzyme catalyzes the reaction that transfers a phosphate group from ATP to fructose-6 phosphate (F6P) to form fructose-1,6 bisphosphate (F16BP) and ADP. Although ATP is a substrate for this reaction and is the source of the transferred phosphate, it also serves as an inhibitor of the enzyme (Kemp and Krebs, 1967 and, ultimately, ATP production. Large amounts of ADP in the cell indicate a lack of energy; thus, ADP activates PFK.…”

Section: Introductionmentioning

confidence: 99%

Effects of oral administration of sodium citrate or acetate to pigs on blood parameters, postmortem glycolysis, muscle pH decline, and quality attributes of pork1,2

Stephens

Dikeman

Unruh

et al. 2008

Journal of Animal Science

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The objective of this study was to determine the effects of oral administration of sodium citrate (CIT) or acetate (ACE) to pigs on blood parameters, postmortem glycolysis, pH decline, and quality attributes of pork. Previous studies have shown that CIT has the potential to inhibit phosphofructokinase (PFK), a key enzyme in postmortem muscle glycolysis. In Exp. 1, CIT, ACE, or water was orally administered (0.75 g/ kg of BW) to 24 pigs. After a 30-min rest, pigs were exercised, and blood samples were taken at 45 and 75 min after oral treatment. Citrate and ACE tended (P = 0.08) to increase blood pH and increased (P = 0.02) bicarbonate levels immediately after exercise. After a 30-min rest, blood pH of pigs administered ACE tended (P = 0.09) to remain higher, whereas blood pH of CITtreated pigs was similar to that of control pigs. Bicarbonate levels in ACE-and CIT-treated pigs were still

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“…With a total of seven signals from the adenine nucleotide pool, a combined response by an enzyme to several of these is one of the methods by which an increased sensitivity and sharpened response to the state of the pool can be achieved, as is exemplified by phosphofructokinase ; although the precise effects of all these species have not been elucidated, the following is probably reasonably accurate : there is a requirement for magnesium as a cofactor in addition to the requirement for magnesium to form the substrate MgATP2-[24,25] ; ATP4-inhibits the binding of fructose-6-phosphate, and this inhibition is released by inorganic phosphate [26]; MgATP2-may also inhibit the enzyme [27], but a M e r e n t mechanism is indicated [26] ; the binding of fructose-6-phosphate is increased in the presence of AMP2-and ADP3-, which compete for a single binding site [26]. When considered in conjunction with the data in the tables, these effects are seen to combine to inhibit phosphofructokinase a t high values of the "energy charge", and to activate the enzyme as the "energy charge" is lowered.…”

Section: Magnesium and The Composition Of An Adeninementioning

confidence: 99%

Magnesium, Potassium, and the Adenylate Kinase Equilibrium

Blair

1970

European Journal of Biochemistry

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The influence of magnesium and potassium on the equilibrium catalysed by adenylate kinase has been studied. An equation has been derived which describes the effect of these ions on the equilibrium; values for the metal‐nucleotide stability constants, and for the “true” equilibrium constant, [AMP2−] · [ATP4−]/([ADP3−])2, have been calculated, which give the best fit of this equation to the experimental data. Using these constants, the concentrations of all significant species in an adenine nucleotide pool equilibrated by adenylate kinase can be calculated, for any specified total concentrations of magnesium, potassium, and adenine nucleotide pool, at any pH and any pyrophosphate bond content of the adenine nucleotide pool. Magnesium emerges as a significant feedback signal from an adenine nucleotide pool considered as an isolated system. The possible significance of this data with respect to the control of metabolic flux in general and of phosphofructokinase in particular is discussed.

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Binding of Metabolites by Phosphofructokinase^*

Cited by 169 publications

References 20 publications

Magnetic Resonance Studies on Manganese‐Activated Phosphofructokinase

Magnetic Resonance Studies on Manganese‐Activated Phosphofructokinase

Effects of oral administration of sodium citrate or acetate to pigs on blood parameters, postmortem glycolysis, muscle pH decline, and quality attributes of pork1,2

Magnesium, Potassium, and the Adenylate Kinase Equilibrium

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Binding of Metabolites by Phosphofructokinase*

Cited by 169 publications

References 20 publications

Magnetic Resonance Studies on Manganese‐Activated Phosphofructokinase

Magnetic Resonance Studies on Manganese‐Activated Phosphofructokinase

Effects of oral administration of sodium citrate or acetate to pigs on blood parameters, postmortem glycolysis, muscle pH decline, and quality attributes of pork1,2

Magnesium, Potassium, and the Adenylate Kinase Equilibrium

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Binding of Metabolites by Phosphofructokinase^*