Kinetic studies on the regulation of rabbit liver pyruvate kinase

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K(+) and optimum activity was recorded with 30mm-K(+), 4mm-MgADP(-), with a MgADP(-)/ADP(2-) ratio of 50:1, but inhibition occurred with K(+) concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg(2+) was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (n(H)=2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent K(m) for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis-Menten response was obtained when phosphoenolpyruvate was the variable substrate (K(m)=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg(2+).     

Studies on the interaction between rabbit liver pyruvate kinase and its allosteric effector fructose 1,6-diphosphate

Preparation of the L form of rabbit liver pyruvate kinase (EC 2.7.1.40) in the presence of fructose 1,6-diphosphate yielded an enzyme which was kinetically identical with the M or muscle-type form of pyruvate kinase found in liver. Chromatographic and dialysis studies of this complex showed that most of the fructose 1,6-diphosphate molecules were loosely bound to the enzyme, but dilution-dissociation studies and binding experiments established that there was a high initial affinity between the enzyme and fructose 1,6-diphosphate (K(assoc.)=2.3x10(9)), and that binding of the loosely bound fructose 1,6-diphosphate was concentration-dependent and a necessary condition to overcome the co-operative interaction observed with the homotropic effector phosphoenolpyruvate. Preparation of the liver enzyme in the absence of EDTA did not yield a predominantly M form of the enzyme, and incubation of the M form in the presence of EDTA did not convert it into the L form, but resulted in inhibition of enzyme activity. Immunological studies confirmed that the L and M forms in liver were distinct, and that preparation of the L form in the presence of fructose 1,6-diphosphate did not produce an enzyme antigenically different from the L form prepared in the absence of this heterotropic effector.     

Regulation of rat liver pyruvate kinase. The effect of preincubation, pH, copper ions, fructose 1,6-diphosphate and dietary changes on enzyme activity

1. Preincubation of partially purified rat liver L-type pyruvate kinase at 25 degrees for 10min. causes a marked increase in co-operativity with respect to both the substrate, phosphoenolpyruvate, and the allosteric activator, fructose 1,6-diphosphate. 2. The results are consistent with the existence of two forms of liver L-type pyruvate kinase, designated forms L(A) and L(B). It is postulated that form L(A) has a low K(m) for phosphoenolpyruvate (about 0.1mm) and is not allosterically activated, whereas form L(B) is allosterically activated by fructose 1,6-diphosphate, exhibiting in the absence of the activator sigmoidal kinetics with half-maximal activity at about 1mm-phosphoenolpyruvate. In the presence of fructose 1,6-diphosphate, form L(B) gives Michaelis-Menten kinetics with K(m) less than 0.1mm. It is further postulated that preincubation converts form L(A) into form L(B). 3. The influence of pH on the preincubation effect was studied. 4. The inhibition of pyruvate kinase by Cu(2+) was studied in detail. Though phosphoenolpyruvate and fructose 1,6-diphosphate readily protect the enzyme against Cu(2+) inhibition, little evidence of significant reversal of the inhibition by these compounds could be found. 5. The effects of starvation, fructose feeding and preincubation on the pyruvate kinase activity of crude homogenates of various tissues of the rat were also studied.     

Kinetic properties of rat liver pyruvate kinase at cellular concentrations of enzyme, substrates and modifiers

Kinetic properties of rat liver pyruvate kinase type I at pH7.5 and 6.5 were studied with physiological ranges of substrates, modifiers and Mg(2+) concentrations at increasing enzyme concentrations, including the estimated cellular concentrations (approx. 0.1mg/ml). Enzyme properties appear unaffected by increased enzyme concentration if phosphoenolpyruvate, fructose 1,6-diphosphate and inhibitors are incubated with enzyme before starting the reaction with ADP. Our data suggest that minimum cellular concentrations of MgATP and l-alanine provide virtually complete inhibition of pyruvate kinase I at pH7.5. The most likely cellular control of existing pyruvate kinase I results from the strong restoration of enzyme activity by the small physiological amounts of fructose 1,6-diphosphate. Decreasing the pH to 6.5 also restores pyruvate kinase activity, but to only about one-third of its activity in the presence of fructose 1,6-diphosphate. Neither pyruvate nor 2-phosphoglycerate at cellular concentrations inhibit the enzyme significantly.     

Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3', 5'-AMP-stimulated protein kinase.

The kinetics of rat liver L-type pyruvate kinase (EC 2.7.1.40), phosphorylated with cyclic AMP-stimulated protein kinase from the same source, and the unphosphorylated enzyme have been compared. The effects of pH and various concentrations of substrates, Mg2+, K+ and modifiers were studied. In the absence of fructose 1, 6-diphosphate at pH 7.3, the phosphorylated pyruvate kinase appeared to have a lower affinity for phosphoenolpyruvate (K0.5=0.8 mM) than the unphosphorylated enzyme (K0.5=0.3 mM). The enzyme activity vs. phosphoenolpyruvate concentration curve was more sigmoidal for the phosphorylated enzyme with a Hill coefficient of 2.6 compared to 1.6 for the unphosphorylated enzyme. Fructose 1, 6-diphosphate increased the apparent affinity of both enzyme forms for phosphoenolpyruvate. At saturating concentrations of this activator, the kinetics of both enzyme forms were transformed to approximately the same hyperbolic curve, with a Hill coefficient of 1.0 and K0.5 of about 0.04 mM for phosphoenolpyruvate. The apparent affinity of the enzyme for fructose 1, 6-diphosphate was high at 0.2 mM phosphoenolpyruvate with a K0.5=0.06 muM for the unphosphorylated pyruvate kinase and 0.13 muM for the phosphorylated enzyme. However, in the presence of 0.5 mM alanine plus 1.5 mM ATP, a higher fructose 1, 6-diphosphate concentration was needed for activation, with K0.5 of 0.4 muM for the unphosphorylated enzyme and of 1.4 muM for the phosphorylated enzyme. The results obtained strongly indicate that phosphorylation of pyruvate kinase may also inhibit the enzyme in vivo. Such an inhibition should be important during gluconeogenesis.     

Factors affecting the activity of pyruvate kinase of Acetobacter xylinum

1. Extracts of Acetobacter xylinum were found to contain the glycolytic enzymes involved in the conversion of triose phosphate into pyruvate. Pyruvate kinase had the lowest relative activity. Phosphofructokinase activity was not detected in the extracts. 2. Only slight differences in the activity of pyruvate kinase were observed between cells grown on glucose and those grown on intermediates of the tricarboxylic acid cycle. 3. Pyruvate kinase, partially purified from ultrasonic extracts by ammonium sulphate fractionation, required Mg(2+) ions for activity. It was not activated by K(+) or NH(4) (+) ions. 4. The plots representing the relationship between initial velocity and phosphoenolpyruvate concentration were sigmoidal, suggesting a co-operative effect for phosphoenolpyruvate. The Hill coefficient (n) for phosphoenolpyruvate was 2. The rate of the reaction changed with increasing ADP concentrations according to normal Michaelis-Menten kinetics. 5. The enzyme was inhibited by ATP (K(i)0.9x10(-3)m). The inhibition was competitive with regard to ADP but not with regard to phosphoenolpyruvate. It was not relieved by excess of Mg(2+) ions. 6. The possible relationship of the properties of pyruvate kinase to regulatory mechanisms for controlling gluconeogenesis and carbohydrate oxidation in A. xylinum is discussed.     

A kinetic study of baker''s-yeast pyruvate kinase activated by fructose 1,6-diphosphate

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by yeast pyruvate kinase when activated by fructose 1,6-diphosphate and K(+). The experimental results indicate that the reaction mechanism is of the Ordered Tri Bi type with the substrates binding in the order phosphoenolpyruvate, ADP and Mg(2+). Direct phosphoryl transfer takes place in the quaternary complex, with pyruvate released before MgATP. A dead-end enzyme-pyruvate complex is also indicated. Values have been determined for the Michaelis, dissociation and inhibition constants of the reaction. Several of the rate constants involved have also been evaluated.     

Long-term effect of glucagon administration on rat liver L-type pyruvate kinase.

After 5 h of treatment with glucagon, liver L-type pyruvate kinase (ATP: pyruvate 2-0-phosphotransferase; EC 2.7.1.40) showed a significant decrease of K0.5 and the Hill coefficient (nH) in the absence of fructose 1,6-diphosphate. However, in the presence of fructose 1,6-diphosphate, liver enzymes from treated rats showed a slight decrease of K0.5 but nH remained unchanged. In both circumstances, no changes of Vmax were observed after treatment. These changes in the kinetic properties of liver L-type pyruvate kinase are consistent with the dephosphorylation of the enzyme caused by insulin release in response to treatment with glucagon.     

Purification of four pyruvate kinase isozymes of rats by affinity elution chromatography.

1. Purification of four isozymes of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) L, M1, M2 and R was much improved to give good yields by affinity elution chromatography. The enzyme was eluted from a phosphocellulose column with 0.5 mM phosphoenolpyruvate. Types L, M2 and R were stabilized with fructose 1,6-diphosphate throughout the purification procedures. 2. The isozymes were crystallized under various conditions: types L and R were readily crystallized from medium of low ionic strength, types L, M1, and M2 were crystallized from ammonium sulfate solution in different forms in the presence and absence of phosphoenolpyruvate. Type M1 was also crystallized in different forms in the presence and absence of fructose 1,6-diphosphate. 3. Amino acid analyses showed that the compositions of types L and R, and of types M1 and M2, respectively, were very similar.     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Functional changes associated with the sequential transformation of L'4 into L4 pyruvate kinase.

The functional changes, associated with the sequential transformation of L'4 into L4 pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) were studied. L'4 enzyme from human erythrocytes shows strong hysteretic behaviour: the initial rate of the enzyme preincubated with an unsaturating concentration of phosphoenolpyruvate is much higher than of the enzyme preincubated with ADP, at the same phosphoenolpyruvate concentration, although the "final activity" (the activity of the linear part of the reaction progress curve) was the same in both cases. This phenomenon was observed both in the presence and absence of fructose 1,6-diphosphate. High concentrations of both Mg2+free and MgATP2- diminish the difference in initial rate, between the ADP and phosphoenolpyruvate preincubated enzymes: Mg2+free by stabilizing the phosphoenolpyruvate-induced form; ATPMg2- by stabilizing the ADP-induced form. The magnitude of the difference in initial rates of the ADP-or phosphoenolpyruvate-preincubated enzyme is a function of both substrates. L4 pyruvate kinase (either from human liver or trypsin treated L'4 enzyme) does not, or to a very slight extent, show such behaviour. L'2L2 pyruvate kinase shows behaviour intermediate between L'4 and L4 enzymes. A model is proposed to describe the kinetic behaviour of L'4 and L4 enzymes.     

An investigation of the interactions of the allosteric modifiers of pyruvate kinase with the enzyme from Carcinus maenas hepatopancreas.

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.     

Temperature and the regulation of enzyme activity in poikilotherms. Regulatory properties of fructose diphosphatase from muscle of the Alaskan king-crab

1. The properties of fructose diphosphatase from skeletal muscle of the Alaskan king-crab (Paralithodes camtschatica) were examined over the physiological temperature range of the animal. 2. King-crab muscle fructose diphosphatase is first activated by Na(+) and NH(4) (+) and is then partially inhibited by these cations at concentrations higher than 10mm at 0 degrees , 8 degrees and 15 degrees C. Enzyme activity is stimulated by K(+) at 0 degrees C, but is curtailed at 8 degrees C and 15 degrees C, an effect that could render rate independent of temperature. 3. Affinity for substrate increases with decreasing temperature; below the temperature of acclimatization, K(m) for fructose 1,6-diphosphate increases, resulting in a complex U-shaped temperature-K(m) curve. 4. King-crab muscle fructose diphosphatase is inhibited by low concentrations of AMP. As with enzymes of other poikilotherms, inhibition by AMP is sensitive to temperature; the enzyme is least sensitive to inhibition by AMP near the temperature of acclimatization. 5. The affinity of fructose diphosphatase for fructose 1,6-diphosphate is enhanced by phosphoenolpyruvate, and this activation is temperature-sensitive; 0.5mm-phosphoenolpyruvate causes a sevenfold decrease in K(m) for fructose 1,6-diphosphate at 15 degrees C but a 25-fold decrease at 0 degrees C. 6. Phosphoenolpyruvate appears to decrease the affinity of king-crab muscle fructose diphosphatase for AMP at low temperature, whereas at the higher temperature it appears to enhance inhibition by AMP. Phosphoenolpyruvate was not observed to cause a reversal of inhibition of fructose diphosphatase activity by AMP. The identification of phosphoenolpyruvate as an activator of a rate-limiting step in gluconeogenesis permits the suggestion of a coupling of the controlling mechanisms of several steps in the glycolytic and gluconeogenic chains. 7. These findings suggest mechanisms for the maintenance and regulation of control of fructose diphosphatase activity in king-crab skeletal muscle at low temperature and under conditions that favour concomitant activity of phosphofructokinase.     

Post-mortem glycolysis in ox skeletal muscle. Effect of pre-rigor freezing and thawing on the intermediary metabolism

1. Ox sternomandibularis muscle was ;slow-frozen' by placing it in air at -22 degrees or ;fast-frozen' by immersion in liquid air or acetone-solid carbon dioxide. In all cases muscles were frozen pre-rigor. Changes in length, pH and the concentrations of P(i), creatine phosphate, hexose monophosphate (glucose 1-phosphate+glucose 6-phosphate+fructose 6-phosphate), fructose diphosphate (fructose 1,6-diphosphate+(1/2) triose phosphate), lactate, ATP, ADP, AMP and NAD(+) during freezing and during subsequent thawing were determined. In addition some measurements were made of the changes in alpha-glycerophosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate and pyruvate concentrations during slow freezing. 2. Appreciable shortening and marked changes in chemical composition took place during slow freezing but not during fast freezing. 3. During slow freezing the hexose monophosphate concentration fell and fructose 1,6-diphosphate and triose phosphate increased substantially. Increases also took place in 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate, but not in pyruvate. 4. On thawing, most of the chemical changes were similar to those in unfrozen muscle post mortem, but took place much more rapidly; loss of NAD(+) was particularly rapid. Fast-frozen muscle metabolized at a faster rate on thawing than did slow-frozen muscle. 5. The overall changes in length during freezing and thawing were about the same in slow-frozen as in fast-frozen muscle.     

Kinetic properties of cerebral pyruvate kinase

Partly purified guinea-pig brain pyruvate kinase is not activated by fructose 1,6-diphosphate and gives hyperbolic substrate-saturation curves with phosphoenolpyruvate. It is therefore different from the L-type pyruvate kinase of mammalian liver. Inhibition by MgATP(2-) was competitive for MgADP(-) but not for phosphoenolpyruvate, and the enzyme is therefore different from the M-type pyruvate kinase, which is said to be competitively inhibited by MgATP(2-) with respect to both substrates. The K(i)(MgATP(2-)) value of approx. 8mm for the brain enzyme is higher than the values (about 2mm) reported for the muscle enzyme. Stimulation of enzymic activity was observed at low (1-2mm) concentrations of MgATP(2-). Substrate kinetic constants were K(m) (MgADP(-))=0.47mm, K(m) (phosphoenolpyruvate)=0.08mm. Free Mg(2+) at very high concentrations (over 10mm) was inhibitory (K(i)=20-32mm). Neither ADP(3-) nor 5'-AMP(2-) inhibited the activity. The brain enzyme was concluded to be different from both the M-type and the L-type of other mammalian organs such as muscle and liver.     

Reversal of glycolysis in yeast.

When a buffered, aerobic suspension of ethanol-grown cells of Saccharomyces cerevisiae is treated with ethanol, a rapid flux of metabolism is observed from endogenous phosphoenolpyruvate to hexose monophosphates. Intracellular concentrations of phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate record a monotonic drop, while those of triose phosphates and fructose 1,6-diphosphate fall after an early rise; fructose 6-phosphate, mannose 6-phosphate, and glucose 6-phosphate levels rise to a plateau. Prior growth on glucose extinguishes fructose 1,6-diphosphatase activity and completely arrests the rise of the hexose monophosphates. By using mutants blocked at a number of glycolytic steps it has been concluded that the metabolic flow takes place along the Embden-Meyerhof pathway in the reverse direction bypassing pyruvate kinase and fructose 6-phosphate kinase. Ethanol acts as a trigger by supplying NADH at the glyceraldehyde 3-phosphate dehydrogenase step. The rate of the reversal in the span phosphoenolpyruvate to fructose 1,6-diphosphate approaches 40 μ mol of 3-carbon units per minute per gram of wet cells. The in vivo activity of fructose 1,6-diphosphatase is nearly a quarter of this rate.     

Purification and characterization of pyruvate kinase from Trypanosoma brucei

Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been partially purified by carboxymethylcellulose chromatography, and gel filtration. The enzyme is unstable in aqueous solution and requires the presence of a thiol protecting reagent as well as glycerol for the maintenance of activity. Dithiothreitol activates as well as stabilizes the enzyme. Phosphoenolpyruvate allosterically activates trypanosome pyruvate kinase whereas hyperbolic kinetics are found when ADP is the variable substrate. Mg²⁺ or Mn²⁺ ions and a monovalent cation are essential for enzyme activity. Fructose 1,6-diphosphate acts as a heterotropic allosteric activator, markedly decreasing the S_0.5 value for phosphoenolpyruvate from 1.34 to 0.25 mm at 1 mm fructose 1,6-diphosphate and transforms the phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form. The enzyme has a pH optimum of 6.5–7.0 and a molecular weight of 270,000 ± 27,000 as estimated by gel chromatography. Purine nucleotides are the preferred coenzymes for the reaction, having much lower K_m values than the pyrimidine nucleotides. The possible role of pyruvate kinase in the regulation of glycolysis in T. brucei is discussed.     

A comparison of the properties of the pyruvate kinases of the fat body and flight muscle of the adult male desert locust

1. The pyruvate kinases of the desert locust fat body and flight muscle were partially purified by ammonium sulphate fractionation. 2. The fat-body enzyme is allosterically activated by very low (1mum) concentrations of fructose 1,6-diphosphate, whereas the flight-muscle enzyme is unaffected by this metabolite at physiological pH. 3. Flight-muscle pyruvate kinase is activated by preincubation at 25 degrees for 5min., whereas the fat-body enzyme is unaffected by such treatment. 4. Both enzymes require 1-2mm-ADP for maximal activity and are inhibited at higher concentrations. With the fat-body enzyme inhibition by ADP is prevented by the presence of fructose 1,6-diphosphate. 5. Both enzymes are inhibited by ATP, half-maximal inhibition occurring at about 5mm-ATP. With the fat-body enzyme ATP inhibition can be reversed by fructose 1,6-diphosphate. 6. The fat-body enzyme exhibits maximal activity at about pH7.2 and the activity decreases rapidly above this pH. This inactivation at high pH is not observed in the presence of fructose 1,6-diphosphate, i.e. maximum stimulating effects of fructose 1,6-diphosphate are observed at high pH. The flight-muscle enzyme exhibits two optima, one at about pH7.2 as with the fat-body enzyme and the other at about pH8.5. Stimulation of the enzyme activity by fructose 1,6-diphosphate was observed at pH8.5 and above.     

Kinetics of the activation of rat liver pyruvate kinase by fructose 1,6-disphosphate and methods for characterizing hysteretic transitions

1. Kinetics of fructose 1,6-diphosphate activation of liver pyruvate kinase type I inhibited with MgATP and l-alanine are described as a function of enzyme and fructose 1,6-diphosphate concentrations. These results can be explained by a single pseudo-first-order transition of the enzyme into an active form, independent of the enzyme concentration. This rate constant, k(app.)=0.24s(-1) with 0.02mm-fructose 1,6-diphosphate (t(0.9) approximately 10s where t(0.9) is the time for 90% conversion), is an increasing function of fructose 1,6-diphosphate concentration far beyond that needed to maximally activate enzyme equilibrated with fructose 1,6-diphosphate (about 20mum). 2. The model equations are best analysed with numerical techniques which are described. These techniques are useful in studying similar slow transients frequently observed in stopped-flow studies of enzymes. 3. Shorter transients (t(0.9)=0.5-1.5s) were observed in the kinetic response of the enzyme to the addition of MgATP or phosphoenolpyruvate, but were not further characterized.