The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P_i was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.     

The influence of limiting and non-limiting growth conditions on glucose and maltose metabolism in Lactococcus lactis ssp. lactis strains

Three strains of Lactococcus lactis ssp. lactis, a dairy strain 65.1, a type strain ATCC 19435, and a mutant AS 211, were grown on glucose and on maltose under chemostat conditions. When the culture was shifted from glucose-limiting to non-limiting conditions, the product shifted from mixed acids to lactate. Mixed acids were obtained in all maltose cultures; however, an enhanced lactate formation was observed in 19435 and AS 211. An inorganic-phosphate (P_i)-dependent maltose phosphorylase activity was found to be responsible for the initial conversion of maltose. The activation of maltose phosphorylase by Pi was strain-specific. When growth was on maltose under non-limiting conditions, a correlation was found between high initial maltose phosphorylase and -phosphoglucomutase activities and lactate production. No such correlation was observed in maltose-limited cells. In glucose-grown cells under non-limiting conditions, homo-fermentative lactate formation coincided with high concentrations of fructose 1,6-bisphosphate (Fru1,6P ₂) and pyruvate (Pyr) and low concentrations of phosphoenolpyruvate (PPyr). Under limiting conditions, mixed acid formation coincided with low concentrations of Fru1,6P ₂ and Pyr and high concentrations of PPyr. In maltose-grown cells there was no correlation between intracellular intermediary metabolite concentrations and product formation. Therefore, in addition to intracellular intermediary metabolite concentrations, the product formation on maltose is suggested to be regulated by the transport and initial phosphorylating steps.     

Acidic proteome of growing and resting Lactococcus lactis metabolizing maltose

The acidic proteome of Lactococcus lactis grown anaerobically was compared for three different growth conditions: cells growing on maltose, resting cells metabolizing maltose, and cells growing on glucose. In maltose metabolizing cells several proteins were up-regulated compared with glucose metabolizing cells, however only some of the up-regulated proteins had apparent relation to maltose metabolism. Cells growing on maltose produced formate, acetate and ethanol in addition to lactate, whereas resting cells metabolizing maltose and cells growing on glucose produced only lactate. Increased levels of alcohol-acetaldehyde dehydrogenase (ADH) and phosphate acetyltransferase (PTA) in maltose-growing cells compared with glucose-growing cells coincided with formation of mixed acids in maltose-growing cells. The resting cells did not grow due to lack of an amino acid source and fermented maltose with lactate as the sole product, although ADH and PTA were present at high levels. The maltose consumption rate was approximately three times lower in resting cells than in exponentially growing cells. However, the enzyme levels in resting and growing cells metabolizing maltose were similar, which indicates that the difference in product formation in this case is due to regulation at the enzyme level. The levels of 30S ribosomal proteins S1 and S2 increased with increasing growth rate for resting cells metabolizing maltose, maltose-growing cells and glucose-growing cells. A modified form of HPr was synthesized under amino acid starvation. This is suggested to be due to alanine misincorporation for valine, which L. lactis is auxotrophic for. L. lactis conserves the protein profile to a high extent, even after prolonged amino acid starvation, so that the protein expression profile of the bacterium remains almost invariant.     

Characterization of three lactic acid bacteria and their isogenic ldh deletion mutants shows optimization for YATP (cell mass produced per mole of ATP) at their physiological pHs

Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and on the pH for E. faecalis. In S. pyogenes and L. lactis, a change in pH resulted in a clear change in Y(ATP) (cell mass produced per mole of ATP). The pH that showed the highest Y(ATP) corresponded to the pH of the natural habitat of the organisms.     

Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR.

The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait 76, 33-40] was studied in a noninvasive manner by 13C-NMR. The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1-13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild-type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration. Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed.     

Use of 31P nuclear magnetic resonance spectroscopy and 14C fluorography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis

High-resolution 31P nuclear magnetic resonance spectroscopy and 14C fluorography have been used to identify and quantitate intermediates of the Embden-Meyerhof pathway in intact cells and cell extracts of Streptococcus lactis. Glycolysing cells contained high levels of fructose 1,6-bisphosphate (a positive effector of pyruvate kinase) but comparatively low concentrations of other glycolytic metabolites. By contrast, starved organisms contained only high levels of 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate. The concentration of Pi (a negative effector of pyruvate kinase) in starved cells was fourfold greater than that maintained by glycolysing cells. The following result suggest that retention of the phosphoenolpyruvate pool by starved cells is a consequence of Pi-mediated inhibition of pyruvate kinase: the increase in the phosphoenolpyruvate pool (and Pi) preceded depletion of fructose 1,6-bisphosphate, and reduction in intracellular Pi (by a maltose-plus-arginine phosphate trap) caused the restoration of pyruvate kinase activity in starved cells. Time course studies showed that Pi was conserved by formation of fructose 1,6-bisphosphate during glycolysis. Conversely, during starvation high levels of Pi were generated concomitant with depletion of intracellular fructose 1,6-bisphosphate. The concentrations of Pi and fructose 1,6-bisphosphate present in starved and glycolysing cells of S. lactis varied inversely. The activity of pyruvate kinase in the growing cell may be modulated by the relative concentrations of the two antagonistic effectors.     

Effect of fructose 2,6-bisphosphate on the kinetic properties of cytoplasmic fructose 1,6-bisphosphatase from germinating castor bean endosperm

The cytoplasmic form of fructose 1,6-bisphosphatase (FBPase) was purified over 60-fold from germinating castor bean endosperm (Ricinus communis). The kinetic properties of the purified enzyme were studied. The preparation was specific for fructose 1,6-bisphosphate and exhibited optimum activity at pH 7.5. The affinity of the enzyme for fructose 1,6-bisphosphate was reduced by AMP, which was a mixed linear inhibitor. Fructose 2,6-bisphosphate also inhibited FBPase and induced a sigmoid response to fructose 1,6-bisphosphate. The effects of fructose 2,6-bisphosphate were enhanced by low levels of AMP. The latter two compounds interacted synergistically in inhibiting FBPase, and their interaction was enhanced by phosphate which, by itself, had little effect. The enzyme was also inhibited by ADP, ATP, UDP and, to a lesser extent, phosphoenolpyruvate. There was no apparent synergism between UDP, a mixed inhibitor, and fructose 2,6-bisphosphate. Similarly ADP, a predominantly competitive inhibitor, did not interact with fructose 2,6-bisphosphate. Possible roles for fructose 2,6-bisphosphate and the other effectors in regulating FBPase are discussed.     

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.     

Study on the initial kinetics of yeast phosphofructokinase by stopped-flow measurements

The initial kinetics of yeast phosphofructokinase was studied by stopped-flow measurements over an enzyme concentration range from 0.5 mg/ml to 0.01 mg/ml. Before attaining the steady state the reaction showed a lag phase in the product formation, the duration of which was found to decrease with increasing enzyme concentration. The lag phase disappeared after preincubation of the enzyme for at least five minutes with either fructose 6-phosphate, fructose 1,6-bisphosphate or fructose 2,6-bisphosphate. Preincubation of the enzyme with either AMP or ADP resulted in a reduction of this phase, while ATP was without effect. Simultaneous addition of fructose 1,6-bisphosphate to the reaction mixture of the enzyme causes a significant shortening of the transient phase, whereas micromolar concentrations of fructose 2,6-bisphosphate are capable of abolishing the lag phase completely. The occurrence of an initial transient phase suggests that the enzyme after starting the reaction converts from a state of low activity to one of high activity. This conversion mainly depends on the concentration of fructose 1,6-bisphosphate generated in the course of the reaction. In addition an association reaction of the enzyme seems to be involved in the process of conversion of the phosphofructokinase during the initial transient phase.     

The las enzymes control pyruvate metabolism in Lactococcus lactis during growth on maltose

The fermentation pattern of Lactococcus lactis with altered activities of the las enzymes was examined on maltose. The wild type converted 65% of the maltose to mixed acids. An increase in phosphofructokinase or lactate dehydrogenase expression shifted the fermentation towards homolactic fermentation, and with a high level of expression of the las operon the fermentation was homolactic.     

Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio.

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.     

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.     

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis.

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.     

The effect of pH on the interaction of substrates and effector to yeast and rabbit muscle pyruvate kinase

The interaction of fructose 1,6-bisphosphate, phosphoenolpyruvate and ADP with pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from yeast and rabbit muscle has been studied as a function of pH utilizing the quenching of protein fluorescence at 330 nm by these ligands. Both the muscle and the yeast pyruvate kinase interact with either ADP or phosphoenolpyruvate with similar affinity, indicating that the substrate-binding sites for these two isozymes are similar. The major difference between the yeast and muscle isozymes is their affinity with fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate interacts with the yeast isozyme in orders of magnitude more strongly than with the muscle isozyme. Moreover, the affinity of fructose 1,6-bisphosphate to the yeast isozyme is strongly pH-dependent, while the interaction of fructose 1,6-bisphosphate with the muscle isozyme is independent of pH. The data indicate that yeast pyruvate kinase undergoes a conformational change as the pH is increased from 6.0 to 8.5.     

A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas.

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.     

Metabolic control of hepatic gluconeogenesis during exercise.

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.     

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.     

Kinetics of activation of L-lactate dehydrogenase from Streptococcus lactis by fructose 1,6-bisphosphate

A lag is observed before the steady state during pyruvate reduction catalysed by lactate dehydrogenase from Streptococcus lactis. The lag is abolished by preincubation of enzyme with the activator fructose 1,6-bisphosphate before mixing with the substrates. The rate constants for the lag phase showed a linear dependence on fructose-1,6-bisphosphate concentration, with a second-order rate constant of 2.0 X 10(4) M-1 s-1, but were independent of enzyme concentration. Binding of fructose 1,6-bisphosphate produces a decrease in the protein fluorescence of the enzyme. The second-order rate constant for the fluorescence change is twice that for the lag in pyruvate reduction. The results suggest that binding of fructose 1,6-bisphosphate induces a conformational change in the enzyme, producing a form with reduced protein fluorescence and increased activity towards pyruvate reduction.     

Effects of fructose 1,6-bisphosphate on the activation of yeast phosphofructokinase by fructose 2,6-bisphosphate and AMP

Fructose 1,6-bisphosphate decreases the activation of yeast 6-phosphofructokinase (ATP:fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) by fructose 2,6-bisphosphate, especially at cellular substrate concentrations. AMP activation of the enzyme is not influenced by fructose 1,6-bisphosphate. Inorganic phosphate increases the activation by fructose 2,6-bisphosphate and augments the deactivation of the fructose 2,6-bisphosphate activated enzyme by fructose 1,6-bisphosphate. Because various states of yeast glucose metabolism differ in the levels of the two fructose bisphosphates, the observed interactions might be of regulatory significance.     

Interaction of calmodulin with muscle phosphofructokinase. Interplay with metabolic effectors of the enzyme under physiological conditions

The hysteretic calmodulin-induced inactivation of muscle phosphofructokinase and the calmodulin-mediated reactivation are essentially dependent on environmental conditions. The interplay of calmodulin during these reactions and at allosteric conditions with Mg . ATP, fructose 6-phosphate, adenosine 5'-[beta, gamma-imido]triphosphate and with the allosteric effectors AMP, ADP, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate was studied by two techniques. (a) A two-step technique with a preincubation of enzyme, calmodulin and effectors in close to physiological concentrations before dilution into an optimal activity assay. It reveals aggregation and slowly reversible conformation changes. (b) A direct assay of dilute enzyme at allosteric conditions. Dominating in the interplay of calmodulin with metabolic effectors is the competitive-like action of calmodulin on Mg . ATP binding to the regulatory sites of the enzyme. At high enzyme concentrations in the absence of hexose phosphates, i.e. at noncatalytic conditions calmodulin counteracts the stabilization of the highly active tetrameric form caused by Mg . ATP. In the allosteric assay it counteracts the ATP-induced allosteric inhibition. In both cases calmodulin acts synergistic with AMP and ADP. To a minor degree calmodulin also counteracts the stabilization of the tetrameric form caused by fructose 6-phosphate and hexose bisphosphate, now however antagonistically to AMP and ADP. By the demonstrated interactions the enzyme can be slowly and hysteretically shifted between an active tetrameric and an inactive dimeric state under control metabolic conditions and of Ca2+ and calmodulin. Resting conditions will inactivate and high contractile activity reactivate available enzyme.