The mechanism of rabbit muscle phosphofructokinase at pH8.
ABSTRACT: 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 4 mM free Mg2+. Most observations were made at I0.2. 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. 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. 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 ping-pong mechanism, and it is concluded that the reaction is sequential and that dissociation of products is random. 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. The observations on phosphofructokinase are discussed in relation to methods of measurement and interpretation of flux ratios and in relation to the mechanism of other kinase enzymes.
Project description:1. An investigation of the reaction mechanism of the fructose 1,6-bisphosphate-activated pyruvate kinase isolated from the hepatopancreas of the crab Carcinus maenas was conducted. The enzyme was assayed in the presence of 500 microns-fructose 1,6-bisphosphate, 75 mM-KCl and 8 mM-Mg2+free at 25 degrees C. The results are consistent with a rapid-equilibrium random mechanism. 2. Evidence is presented that suggests the formation of two mixed-substrate-product dead-end complexes, enzyme-ADP-pyruvate and enzyme-ADP-ATP. 3. Competitive substrate inhibition was observed for both substrates, ADP and phosphoenolpyruvate, suggesting the formation of the complexes enzyme-ADP-ADP and enzyme-phosphoenolpyruvate-phosphoenolpyruvate in the suggested mechanism. 4. Data from the ATP product-inhibition studies indicate the formation of the complex enzyme-ATP-ATP. This suggests that in the reverse reaction ATP also will show substrate inhibition. 5. The presence of a saturating concentration of fructose 1,6-bisphosphate does not cause full activation of the purified preparations of the enzyme. 6. Pyruvate kinase activity in the supernatant of a hepatopancreas homogenate was completely activated by fructose 1,6-bisphosphate, suggesting that the binding of this ligand to the purified pyruvate kinase was impaired.
Project description:1. Pyruvate kinase purified from the hepatopancrease of Carcinus maenas exhibited sigmoidal saturation kinetics with respect to the substrate phosphoenolpyruvate in the absence of the allosteric activator fructose 1,6-bisphosphate, but normal hyperbolic saturation was seen in the presence of this activator. The activation appears to be the result of a decrease in the s0.5 (phosphoenolpyruvate) and not to a change in Vmax. 2. In the presence of ADP and ATP at a constant nucleotide-pool size the results indicate that phosphoenolpyruvate co-operativity is lost on increasing the [ATP]/[ADP] ratio. 3. Paralleling this change is the observation that the fructose 1,6-bisphosphate activation became less at the [ATP]/[ATP] ratio was increased. This was due to the enzyme exhibiting a near-maximal activity in the absence of activator. 4. L-Alanine inhibited the enzyme, but homotropic co-operative interactions were only seen with a cruder (1000000g supernatant) enzyme preparation. The inhibition by alanine could be overcome by increasing the concentration of either phosphoenolpyruvate or fructose 1,6-bisphosphate, although increasing the L-alanine concentration did not appear to be able to reverse the activation by fructose 1,6-bisphosphate. 5. In the presence of a low concentration of phosphoenolpyruvate, increasing the concentration of the product, ATP, caused an initial increase in enzyme activity, followed by an inhibitory phase. In the presence of either fructose 1,6-bisphosphate or L-alanine only inhibition was seen. 6. The inhibition by ATP could not be completely reversed by fructose 1,6-bisphosphate.
Project description:The steady-state kinetics of the reaction catalysed by the bloodstream form of Trypanosoma brucei were studied at pH 6.7. In the presence of 50 mM-potassium phosphate buffer, the apparent co-operativity with respect to fructose 6-phosphate and the non-linear relationship between initial velocity and enzyme concentration, which were found when the enzyme was assayed in 50 mM-imidazole buffer [Cronin & Tipton (1985) Biochem. J. 227, 113-124], are not evident. Studies on the variations of the initial rate with changing concentrations of MgATP and fructose 6-phosphate, the product inhibition by fructose 1,6-bisphosphate and the effects of the alternative substrate ITP were consistent with an ordered reaction pathway, in which MgATP binds to the enzyme before fructose 6-phosphate, and fructose 1,6-bisphosphate is the first product to dissociate from the ternary complex.
Project description:Prolonged exercise increased the concentrations of the hexose phosphates and phosphoenolpyruvate and depressed those of fructose 1,6-bisphosphate, triose phosphates and pyruvate in the liver of the rat. Since exercise increases gluconeogenic flux, these changes in metabolite concentrations suggest that metabolic control is exerted, at least, at the fructose 6-phosphate/fructose 1,6-bisphosphate and phosphoenolpyruvate/pyruvate substrate cycles. Exercise increased the maximal activities of glucose 6-phosphatase, fructose 1,6-bisphosphatase, pyruvate kinase and pyruvate carboxylase in the liver, but there were no changes in those of glucokinase, 6-phosphofructokinase and phosphoenolpyruvate carboxykinase. Exercise changed the concentrations of several allosteric effectors of the glycolytic or gluconeogenic enzymes in liver; the concentrations of acetyl-CoA, ADP and AMP were increased, whereas those of ATP, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were decreased. The effect of exercise on the phosphorylation-dephosphorylation state of pyruvate kinase was investigated by measuring the activities under conditions of saturating and subsaturating concentrations of substrate. The submaximal activity of pyruvate kinase (0.5 mM-phosphoenolpyruvate), expressed as percentage of Vmax., decreased in the exercised animals to less than half that found in the controls. These changes suggest that hepatic pyruvate kinase is less active during exercise, possibly owing to phosphorylation of the enzyme, and this may play a role in increasing the rate of gluconeogenesis.
Project description:6-Phosphofructokinase purified from honey-bee flight muscle is inhibited by ATP and, unusually, by glucose 1,6-bisphosphate and fructose 1,6-bisphosphate. The inhibition by either of the bisphosphates is not relieved by AMP, but is relieved by fructose 6-phosphate and especially by fructose 2,6-bisphosphate. Lack of effect by AMP is consistent with a low activity of adenylate kinase in this muscle.
Project description:Addition of glucose to Ehrlich-Landschütz ascites tumour cells preincubated for 30-60 min in phosphate-buffered Krebs-Ringer salt solution ("starved cells") resulted within 1-2 min in an approx. 90% decline of their ATP content and a massive accumulation of fructose 1,6-bisphosphate. These alterations, which took place under both aerobic and anaerobic conditions, were followed by a gradual spontaneous recovery with restoration of normal ATP and fructose 1,6-bisphosphate values. The transient derangement of the energy metabolism after glucose addition to starved ascites tumour cells by preventable by simultaneous addition of pyruvate or 2-oxobutyrate, or by preincubating the cells in the presence of glucose. The protective effect of pyruvate was duplicated by addition of phenazine methosulphate or NAD+ to the incubation medium. The data seem to warrant the conclusion that the glucose-induced ATP depletion is determined by a blockade of glycolysis at the stage of glyceraldehyde phosphate dehydrogenase caused by the failure of the cells to oxidize the NADH produced in the same reaction. The continued unrestrained action of 6-phosphofructokinase results in accumulation of fructose 1,6-bisphosphate, which constitutes a trap for the high-energy phosphate bonds of ATP. The primary metabolic disturbance appears to consist of a transient inhibition of pyruvate kinase with the resultant inability of the cells to maintain an unimpaired supply of pyruvate, as required for the lactate dehydrogenase-mediated oxidation of NADH. The regulatory mechanism underlying this phenomenon is discussed.
Project description:Glutamine caused a dose-dependent decrease in fructose 2,6-bisphosphate concentration in both HeLa cells and chick-embryo fibroblasts. The effect was complete within 15 min in HeLa cells, but required more than 9 h in the fibroblasts. Half-maximal effects were obtained with 0.1-0.3 mM-glutamine. In chick-embryo fibroblasts, but not in HeLa cells, glutamine induced a time-dependent decrease in the activity of phosphofructokinase-2, which correlated with the decrease in fructose 2,6-bisphosphate. Glutamine decreased the glycolytic flux by about 25% only in chick-embryo fibroblasts. The difference in glycolytic response between the two types of cells might correspond to a difference in the sensitivity of phosphofructokinase-1 for fructose 2,6-bisphosphate. In HeLa cells, glutamine caused a 2-3-fold stimulation of the synthesis of glycogen, a 50% decrease in the concentration of fructose 1,6-bisphosphate and a more than 80% decrease in the concentration of 5-phosphoribosyl pyrophosphate; the concentrations of hexose 6-phosphates and ATP were not affected.
Project description:Glucose 1,6-bisphosphate, fructose 2,6-bisphosphate, glycogen, lactate and other glycolytic metabolites were measured in rat gastrocnemius muscle, which was electrically stimulated in situ via the sciatic nerve. Both the frequency and the duration of stimulation were varied to obtain different rates of glycolysis. There was no apparent relationship between fructose 2,6-bisphosphate content and lactate accumulation in contracting muscle. In contrast, glucose 1,6-bisphosphate content increased with lactate concentration during contraction. It is suggested that the increase in glucose 1,6-bisphosphate could play a role in phosphofructokinase stimulation and in the activation of the glycolytic flux during muscle contraction.
Project description:Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transporters are both expressed by trypanosomes and these parasites can infiltrate tissues that contain glycerol. Here, we show that bloodstream form trypanosomes can use glycerol for gluconeogenesis and for ATP production, particularly when deprived of glucose following hexose transporter depletion. We demonstrate that Trypanosoma brucei hexose transporters 1 and 2 (THT1 and THT2) are localized to the plasma membrane and that knockdown of THT1 expression leads to a growth defect that is more severe when THT2 is also knocked down. These data are consistent with THT1 and THT2 being the primary routes of glucose supply for the production of ATP by glycolysis. However, supplementation of the growth medium with glycerol substantially rescued the growth defect caused by THT1 and THT2 knockdown. Metabolomic analyses with heavy-isotope labelled glycerol demonstrated that trypanosomes take up glycerol and use it to synthesize intermediates of gluconeogenesis, including fructose 1,6-bisphosphate and hexose 6-phosphates, which feed the pentose phosphate pathway and variant surface glycoprotein biosynthesis. We used Cas9-mediated gene knockout to demonstrate a gluconeogenesis-specific, but fructose-1,6-bisphosphatase (Tb927.9.8720)-independent activity, converting fructose 1,6-bisphosphate into fructose 6-phosphate. In addition, we observed increased flux through the tricarboxylic acid cycle and the succinate shunt. Thus, contrary to prior thinking, gluconeogenesis can operate in bloodstream form T. brucei. This pathway, using glycerol as a physiological substrate, may be required in mammalian host tissues.
Project description:1. Ox heart phosphofructokinase catalyses isotope-exchange reactions at pH6.7 between ADP and ATP, and between fructose 6-phosphate and fructose 1,6-diphosphate, the latter reaction being absolutely dependent on the presence of the magnesium complex of ADP. 2. The reaction kinetics are hyperbolic with respect to substrate concentration for both exchange reactions (within the experimental error). 3. The influence of pH, AMP and citrate suggests that the fructose 6-phosphate-fructose 1,6-diphosphate exchange is subject to effector control, and is abolished by dissociation of the enzyme. 4. These results are discussed in relation to the reaction mechanism of the enzyme.