Interconvertible Forms of Phosphofructokinase of Rabbit Liver

Abstract

When the phosphofructokinase reaction was started with the addition of stock rabbit liver phosphofructokinase its activity declined rapidly, a t low concentrations of fructose 6-phosphate or a t high concentrations of ATP. The rate of this decline is independent of enzyme concentration and it could be prevented by the presence of positive effectors of the enzyme or by high concentration of fructose 6-phosphate.When the reaction rate was measured in the first few seconds after addition of stock enzyme, the activity of the enzyme and its [S],., value (concentration of fructose 6-phosphate for half maximal velocity) were not affected significantly by the presence of positive effectors of the enzyme. The stock enzyme, which was routinely stored in the presence of 0.7M (NH,),SO,, i0mM K,HPO,, 1OmM ATP, 50mM Tris-HC1 pH 7.5 and 0.33mM EDTA, was transformed to a less active form of enzyme, which had a very high [S],., value and low intrinsic activity on dilution or when freed of effectors by passing the enzyme solution through a Sephadex G-25 column. This form can be converted back to the active form by the positive effectors of the enzyme. The rate of this conversion of less active form to the active form of enzymes is again independent of enzyme concentration.The rate-limiting step in the interconversions of active form to less active form of enzyme or vice versa is a first-order process and there appears to be no aggregation or disaggregation of the enzyme during these interconversions.A variety of [S],., values and maximal velocities were observed for the enzyme depending on the preincubation, prior dilution andsemoval of the effectors of enzyme suggesting the existence of a t least two conformational states of enzyme in varying proportions differing widely in their It was shown earlier that partially purified rabbit liver phophofructokinase stored in the presence of NH,+, Kf, SO,,-, Pi and ATP exists in a form with high affinity for fructose 6-phosphate and on dilution is transformed to a form with affinity for fructose 6-phosphate a t pH 7.5 [1,2]. It was suggested that these observations may be interpreted on the basis of slow conformational change( s) or aggregationdisaggregation of the enzyme or both, as a result of changes in the concentration of enzyme and effectors.Enzyme. Phosphofructokinase or ATP : D-fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.11).Definition.[S],.,, the concentration of fructose 6-phosphate required for half maximal velocity under the given conditions of assay.I n this article it is shown that the presence of effectors in the stock enzyme solution keeps the enzyme in the active state with high intrinsic activity and affinity for fructose 6-phosphate, and when these effectors are removed either by dilution or by passing the enzyme through Sephadex G-25, the enzyme is transformed slowly to a form with very low affinity for fructose 6-phosphate and relatively low intrinsic activity. The less active form of enzyme could be converted back to the active form by the positive effectors o...

Rat liver phosphofructokinase: kinetic activity under near-physiological conditions

“…The synergistic effects of the three activators FBP, AMP, and P, suggest that different binding sites exist for each ligand, as is the case for the muscle enzyme (Lowry & Passonneau, 1966). Discussion Ramaiah & Tejwani (1973) observed kinetic transients similar to those described here in their work with rabbit liver PFK and interpreted their results in terms of the allosterism model of Monod et al (1965) where the slow interconversion of two enzyme conformations produced transients. We prefer to interpret these effects as reflecting the binding antagonism between F6P and MgATP, presumably at the active site and allosteric site, respectively.…”

“…Hill coefficients are listed in the inset. limiting step were a conformational change of the protein in the absence of ligands, as proposed by Ramaiah & Tejwani (1973), then one would expect the inhibition of the initial rate, which results from placing "active" stock enzyme in excess of MgATP prior to initiating with F6P, to take as long to occur as the inhibition observed when the reaction is started with ATP. This is not the case, however, as reactions begun with F6P after a very short time of enzyme incubation with MgATP (<1 min) show complete inhibition of the initial rate, whereas burst transients generally have half-times greater than 2 min (Figure 2A).…”

“…Carbohydrate metabolism of cells capable of performing gluconeogenesis, such as liver and kidney, is fundamentally more complex, and the PFK1 isolated from these sources has been found to have, as expected, altered characteristics. Liver isozymes of PFK from a variety of species including rabbit (Kemp, 1971;Ramaiah & Tejwani, 1973) , pig (Massey & Deal, 1973), chicken (Kono & Uyeda, 1974) , sheep (Brock, 1969), and rat (Brand & Soling, 1974;Dunaway & Weber, 1974;Underwood & Newsholme, 1965) have been studied. Since a large majority of the studies of gluconeogenesis, including those in our own laboratory, are performed with the rat, we were interested in more closely investigating the kinetic properties of the liver enzyme from this source.…”

Creation of an Allosteric Phosphofructokinase Starting with a Nonallosteric Enzyme

“…Thus suggesting that both phenomena represent the same process, as shown for regulated PFKs (12,42). The question arises as to how the marked cooperativity in Fru-6-P binding to the mutant enzyme is generated.…”

An activation factor of liver phosphofructokinase.

“…We, as well as others (6,12), have observed nonlinear biphasic rate curves when using the purified enzyme under the assay conditions employed for allosteric kinetics. In the presence of the activation factor, however, the rate of the reaction becomes linear.…”