Divalent metal cation requirements of phosphofructokinase-2 from E. coli. Evidence for a high affinity binding site for Mn

The reaction catalyzed by E. coli Pfk-2 presents a dual-cation requirement. In addition to that chelated by the nucleotide substrate, an activating cation is required to obtain full activity of the enzyme. Only Mn²⁺ and Mg²⁺ can fulfill this role binding to the same activating site but the affinity for Mn²⁺ is 13-fold higher compared to that of Mg²⁺. The role of the E190 residue, present in the highly conserved motif NXXE involved in Mg²⁺ binding, is also evaluated in this behavior. The E190Q mutation drastically diminishes the kinetic affinity of this site for both cations. However, binding studies of free Mn²⁺ and metal–Mant-ATP complex through EPR and FRET experiments between the ATP analog and Trp88, demonstrated that Mn²⁺ as well as the metal–nucleotide complex bind with the same affinity to the wild type and E190Q mutant Pfk-2. These results suggest that this residue exert its role mainly kinetically, probably stabilizing the transition state and that the geometry of metal binding to E190 residue may be crucial to determine the catalytic competence.     

Reversible high affinity inhibition of phosphofructokinase-1 by acyl-CoA: a mechanism integrating glycolytic flux with lipid metabolism

The enzyme phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis and is regulated by a complex array of allosteric effectors that integrate glycolytic flux with cellular bioenergetics. Here, we demonstrate the direct, potent, and reversible inhibition of purified rabbit muscle PFK-1 by low micromolar concentrations of long chain fatty acyl-CoAs (apparent Ki～1 μM). In sharp contrast, short chain acyl-CoAs, palmitoylcarnitine, and palmitic acid in the presence of CoASH were without effect. Remarkably, MgAMP and MgADP but not MgATP protected PFK-1 against inhibition by palmitoyl-CoA indicating that acyl-CoAs regulate PFK-1 activity in concert with cellular high energy phosphate status. Furthermore, incubation of PFK-1 with [1-(14)C]palmitoyl-CoA resulted in robust acylation of the enzyme that was reversible by incubation with acyl-protein thioesterase-1 (APT1). Importantly, APT1 reversed palmitoyl-CoA-mediated inhibition of PFK-1 activity. Mass spectrometric analyses of palmitoylated PFK-1 revealed four sites of acylation, including Cys-114, Cys-170, Cys-351, and Cys-577. PFK-1 in both skeletal muscle extracts and in purified form was inhibited by S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, demonstrating that covalent acylation of PFK-1 was not required for inhibition. Tryptic footprinting suggested that S-hexadecyl-CoA induced a conformational change in PFK-1. Both palmitoyl-CoA and S-hexadecyl-CoA increased the association of PFK-1 with Ca2+/calmodulin, which attenuated the binding of palmitoylated PFK-1 to membrane vesicles. Collectively, these results demonstrate that fatty acyl-CoA modulates phosphofructokinase activity through both covalent and noncovalent interactions to regulate glycolytic flux and enzyme membrane localization via the branch point metabolic node that mediates lipid flux through anabolic and catabolic pathways.     

Kinetic analysis of PPi-dependent phosphofructokinase from Porphyromonas gingivalis

We have previously cloned the gene encoding a pyrophosphate-dependent phosphofructokinase (PFK), designated PgPFK, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In this study, recombinant PgPFK was purified to homogeneity, and biochemically characterized. The apparent K(m) value for fructose 6-phosphate was 2.2 mM, which was approximately 20 times higher than that for fructose 1,6-bisphosphate. The value was significantly greater than any other described PFKs, except for Amycolatopsis methanolica PFK which is proposed to function as a fructose 1,6 bisphosphatase (FBPase). The PgPFK appears to serves as FBPase in this organism. We postulate that this may lead to the gluconeogenic pathways to synthesize the lipopolysaccharides and/or glycoconjugates essential for cell viability.     

Ganglioside Treatment of Streptozotocin-Diabetic Rats Prevents Defective Axonal Transport of 6-Phosphofructokinase Activity

This study measured axonal transport of 6-phosphofructokinase (PFK) and aldolase activities in the sciatic nerves of rats with short-term streptozotocin-induced diabetes. The diabetic rats showed deficits in anterograde (69% of controls; p less than 0.001) and retrograde (33% of controls; p less than 0.01) accumulations of PFK activity as well as its content per unit length of unconstricted sciatic nerve (86% of controls; p less than 0.05). There were no accumulation deficits in aldolase activity in the nerves of the diabetic rats, although the activity per unit length of unconstricted nerve was deficient (81% of controls; p less than 0.05). Treatment of diabetic rats with mixed bovine brain gangliosides (10 mg/kg of body weight/day, i.p.) did not affect the deficit in PFK activity in unconstricted nerve (84% of ganglioside-treated controls; p less than 0.01), but all the other defects in enzyme activities were prevented completely. The diabetic rats also showed a reduction of 7% (p less than 0.01) in sciatic nerve dry weight per unit length, which was prevented by ganglioside treatment. In contrast, the reduced motor nerve conduction velocity, accumulation of polyol pathway metabolites, and depletion of myo-inositol, characteristic of untreated short-term diabetes, were unaffected by ganglioside treatment.     

Filamentous actin and its associated binding proteins are the stimulatory site for 6-phosphofructo-1-kinase association within the membrane of human erythrocytes

Glycolytic enzymes reversibly associate with the human erythrocyte membrane (EM) as part of their regulatory mechanism. The site for this association has been described as the amino terminus of band 3, a transmembrane anion transporter. Binding of glycolytic enzymes to this site is recognized to inhibit glycolysis, since binding inhibits the catalytic activity of these enzymes, including the rate-limiting enzyme 6-phosphofructo-1-kinase (PFK). However, the existence of a putative stimulatory site for glycolytic enzymes within the EM has been proposed. PFK has been described as able to reversibly associate with other proteins, such as microtubules, which inhibit the enzyme, and filamentous actin, which activates the enzyme. Here, it is demonstrated that PFK also binds to actin filaments and its associated binding proteins in the protein meshwork that forms the erythrocyte cytoskeleton. Through fluorescence resonance energy transfer experiments using either confocal microscopy or fluorescence spectroscopy, we show that, within the EM, PFK and actin filaments containing its associated binding proteins are located close enough to propose binding between them. Moreover, specifically blocking PFK binding to band 3 results in an association of the enzyme with the EM that increases the enzyme's catalytic activity. Conversely, disruption of the association between PFK and actin filaments containing its associated binding proteins potentiates the inhibitory action of the EM on the enzyme. Furthermore, it is shown that insulin signaling increases the association of PFK to actin filaments and its associated binding proteins, revealing that this event may play a role on the stimulatory effects of insulin on erythrocyte glycolysis. In summary, the present work presents evidence that filamentous actin and its associated binding proteins are the stimulatory site for PFK within the EM.     

Regulation of the futile cycle of fructose phosphate in sea mussel

Carbohydrate metabolism in mussels shows two phases separated seasonally. During summer and linked to food supply, carbohydrates, mainly glycogen, are accumulated in the mantle tissue. During winter, mantle glycogen decreases concomitantly with an increase in triglyceride synthesis. In spring, after spawning, the animals go in to metabolic rest until the beginning of a new cycle. This cycle is regulated by the futile cycle of fructose phosphate that implicates PFK-1 and FBPase-1 activities. These enzymes and the bifunctional PFK-2/FBPase-2 that regulates the Fru-2,6-P₂ levels, are seasonally modulated by covalent phosphorylation/dephosphorylation mechanisms, as a response to unknown factors. The futile cycle of the fructose phosphates also controls the transition from physiological aerobiosis to hypoxia. The process is independent of the phosphorylation state. In this sense, a pH decrease triggers a small Pasteur effect during the first 24 h of aerial exposure. Variations in the concentration of Fru-2,6-P₂ and AMP are the sole factor responsible for this effect. Longer periods of hypoxia induce a metabolic depression characterized by a decrease in Fru-2,6-P₂ which is hydrolyzed by drop in the pH. In this review, the authors speculate on the two regulation processes.     

Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism

Intercellular communication between oocytes and granulosa cells is essential for normal follicular differentiation and oocyte development. Subtraction hybridization was used to identify genes more highly expressed in cumulus cells than in mural granulosa cells of mouse antral follicles. This screen identified six genes involved in glycolysis: Eno1, Pkm2, Tpi, Aldoa, Ldh1, and Pfkp. When oocytes were microsurgically removed from cumulus cell-oocyte complexes, the isolated cumulus cells exhibited decreased expression levels of genes encoding glycolytic enzymes, glycolysis and activity of the tricarboxylic acid (TCA) cycle. These decreases were prevented by culturing the cumulus cells with paracrine factors secreted by fully grown oocytes. Paracrine factors from fully grown oocytes exhibited greater ability than those from growing oocytes to promote expression of genes encoding glycolytic enzymes and glycolysis in the granulosa cells of preantral follicles. However, neither fully grown nor growing oocytes secreted paracrine factors affecting activity of the TCA cycle. These results indicate that oocytes regulate glycolysis and the TCA cycle in granulosa cells in a manner specific to the population of granulosa cells and to the stage of growth and development of the oocyte. Oocytes control glycolysis in granulosa cells by regulating expression levels of genes encoding glycolytic enzymes. Therefore, mouse oocytes control the intercellular metabolic cooperativity between cumulus cells and oocytes needed for energy production by granulosa cells and required for oocyte and follicular development.     

Measurement of glycolysis reactants by high-throughput solid phase extraction with tandem mass spectrometry: Characterization of pyrophosphate-dependent phosphofructokinase as a case study

Glycolysis is a 10-step metabolic pathway involved in producing cellular energy. Many tumors exhibit accelerated glycolytic rates, and enzymes that participate in this pathway are focal points of cancer research. Here, a novel method for the measurement of glycolysis reactants from in vitro samples is presented. Fast and direct measurement is achieved by an automated system that couples on-line solid phase extraction (SPE) with tandem mass spectrometry (MS/MS). The single analytical method enables multiple reactants to be measured concurrently, sustains a cycle time of 8 s, and permits the measurement of up to 10,000 samples per day. Concentration–response curves were conducted using standards for 10 metabolic intermediates, and the results demonstrate that the detection strategy has excellent sensitivity (average limit of detection = 5.4 nM), dynamic range (nanomolar to micromolar), and linear response (average R² = 0.998). To test the analysis method on reactions, pyrophosphate-dependent phosphofructokinase (PPi–PFK) was used as a model system. Data that corroborate the activation and inhibition of PPi–PFK are presented, and the ways in which SPE–MS/MS simplifies experimental design and interpretation are highlighted. In summary, the method for measuring metabolic intermediates described here demonstrates unprecedented speed, performance, and versatility.