The mechanism of rabbit muscle phosphofructokinase at pH8

Abstract

1. The mechanism of rabbit muscle phosphofructokinase was investigated by measurement of fluxes, isotope trapping and steady-state velocities at pH8 in triethanolamine/HCl buffer with 4mM free Mg2+. Most observations were made at I0.2. 2. The ratio Flux of fructose 1,6-bisphosphate--fructose 6-phosphate/Flux of fructose 1,6-bisphosphate-+ATP at zero ATP concentration increased hyperbolically from unity to about 3.2 as the concentration of fructose 6-phosphate was increased.Similarly, the ratio Flux of fructose 1,6-bisphosphate-*ATP/Flux of fructose 1,6-bisphosphate-fructose 6-phosphate at zero fructose 6-phosphate concentration increased from unity to about 1.4 as the concentration of ATP was increased. The addition of substrates must therefore be random, whatever the other aspects of the reaction. Further, from the plateau values of the ratios, it follows that the substrates dissociate very infrequently from the ternary complex and that at a low substrate concentration 72% of the reaction follows the pathway in which ATP adds first to the enzyme. 3. Isotope-trapping studies with [32P]ATP confirmed that ATP can bind first to the enzyme in rate-limiting step and that dissociation of ATP from the ternary complex is slow in relation to the forward reaction. No isotope trapping of [U-14C]-fructose 6-phosphate could be demonstrated. 4. The ratios Flux of ATP-+fructose 1,6-bisphosphate/Flux of ATP-ADP measured at zero ADP concentration and the reciprocal of the ratio measured at zero fructose 1,6-bisphosphate concentration did not differ significantly from unity. Calculated values for these ratios based on the kinetics of the reverse reaction and assuming ordered dissociations of products or a ping-pong mechanism gave values very significantly greater than unity. These findings exclude an ordered dissociation or a substantial contribution from a pingpong mechanism, and it is concluded that the reaction is sequential and that dissociation of products is random. 5. Rate constants were calculated for the steps in the enzyme reaction. The results indicate a considerable degree of co-operativity in the binding between the two substrates. 6. The observations on phosphofructokinase are discussed in relation to methods of measurement and interpretation of flux ratios and in relation to the mechansim of other kinase enzymes.Phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) catalyses the transfer of the terminal phosphate of ATP to the C-1 hydroxy group of Fru6P to produce Fru(1,6)P2 and ADP. It is an important glycolytic enzyme, particularly because it is subject to metabolic control and its velocity is modified by a Abbreviations used: Fru6P, fructose 6-phosphate; Fru(1,6)P2, fructose 1,6-bisphosphate.

Kinetic studies on the activation of pyrophosphate-dependent phosphofructokinase from mung bean by fructose 2,6-bisphosphate and related compounds

“…Thus, the mechanism appears to be rapid equilibrium random in the absence or presence of F2,6P. The ATP-PFK from rabbit muscle also appears to have a random mechanism at basic pH (Bar-Tana & Cleland, 1974; Merry & Britton, 1985). However, the nonregulated ATP-PFK from Lactobacillus plantarum has been reported to have an ordered mechanism in which the sugar substrate binds first (Simon & Hofer, 1978).…”

A Model of Phosphofructokinase and Glycolytic Oscillations in the Pancreatic β-cell

“…We assume that PFK with respect to its substrates, F6P and MgATP, follows rapid equilibrium random bimolecular kinetics (Merry and Britton, 1985). The enzyme may at physiological conditions be considered to be fully saturated with respect to MgATP (Bosca et al, 1985).…”

Isotope Partitioning with Ascaris suum Phosphofructokinase Is Consistent with an Ordered Kinetic Mechanism

“…The established ordered mechanism for Ascaris PFK is unique for a phosphofructokinase. Both rabbit muscle PFK (Merry & Britton, 1985) and Escherichia coli PFK (Deville-Bonne et al, 1991) have a random kinetic mechanism. The pyrophosphate-dependent enzymes (PP i -PFK) from Propionibacterium freudenreichii (Cho et al, 1988), Entamoeba histolytica (Bertagnolli & Cook, 1984), and Phaseoleus aureus (Bertagnolli et al, 1986) all exhibit a rapid equilibrium random kinetic mechanism.…”