Kinetics of the activation of rat liver pyruvate kinase by fructose 1,6-diphosphate and methods for characterizing hysteretic transitions.
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ABSTRACT:
SUBMITTER: Spivey HO
PROVIDER: S-EPMC1168056 | biostudies-other | 1974 Jul
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC1174261 | biostudies-other
Project description: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.
| S-EPMC1177470 | biostudies-other
Project description:Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1?) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency.Yeast and cancer cells both favor sugar fermentation in aerobic conditions. Here the authors describe a conserved mechanism from yeast to mammals where the glycolysis intermediate fructose-1,6-bisphosphate binds Cdc25/Sos1 and couples increased glycolytic flux to increased Ras proto-oncoprotein activity.
| S-EPMC5640605 | biostudies-literature
Project description:Fructose-1,6-bisphosphate (FBP) aldolase, a glycolytic enzyme, catalyzes the reversible and stereospecific aldol addition of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (d-G3P) by an unresolved mechanism. To afford insight into the molecular determinants of FBP aldolase stereospecificity during aldol addition, a key ternary complex formed by DHAP and d-G3P, comprising 2% of the equilibrium population at physiological pH, was cryotrapped in the active site of Toxoplasma gondii aldolase crystals to high resolution. The growth of T. gondii aldolase crystals in acidic conditions enabled trapping of the ternary complex as a dominant population. The obligate 3(S)-4(R) stereochemistry at the nascent C3-C4 bond of FBP requires a si-face attack by the covalent DHAP nucleophile on the d-G3P aldehyde si-face in the active site. The cis-isomer of the d-G3P aldehyde, representing the dominant population trapped in the ternary complex, would lead to re-face attack on the aldehyde and yield tagatose 1,6-bisphosphate, a competitive inhibitor of the enzyme. We propose that unhindered rotational isomerization by the d-G3P aldehyde moiety in the ternary complex generates the active trans-isomer competent for carbonyl bond activation by active-site residues, thereby enabling si-face attack by the DHAP enamine. C-C bond formation by the cis-isomer is suppressed by hydrogen bonding of the cis-aldehyde carbonyl with the DHAP enamine phosphate dianion through a tetrahedrally coordinated water molecule. The active site geometry further suppresses C-C bond formation with the l-G3P enantiomer of d-G3P. Understanding C-C formation is of fundamental importance in biological reactions and has considerable relevance to biosynthetic reactions in organic chemistry.
| S-EPMC5712624 | biostudies-literature
Project description: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.
| S-EPMC1198828 | biostudies-other
Project description:Pyruvate kinase M2 (PKM2) plays a key role in tumor metabolism and regulates the rate-limiting final step of glycolysis. In tumor cells, there are two allosteric effectors for PKM2: fructose-1,6-bisphosphate (FBP) and serine. However, the relationship between FBP and serine for allosteric regulation of PKM2 is unknown. Here we constructed residue/residue fluctuation correlation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in bound PKM2 is distinctly different from that in the free state, FBP/PKM2, or Ser/PKM2. The community network analysis indicates that the information can freely transfer from the allosteric sites of FBP and serine to the substrate site in bound PKM2, while there exists a bottleneck for information transfer in the network of the free state. Furthermore, the binding free energy between the substrate and PKM2 for bound PKM2 is significantly lower than either of FBP/PKM2 or Ser/PKM2. Thus, a hypothesis of "synergistic allosteric mechanism" is proposed for the allosteric regulation of FBP and serine. This hypothesis was further confirmed by the perturbational and mutational analyses of community networks and binding free energies. Finally, two possible synergistic allosteric pathways of FBP-K433-T459-R461-A109-V71-R73-MG2-OXL and Ser-I47-C49-R73-MG2-OXL were identified based on the shortest path algorithm and were confirmed by the network perturbation analysis. Interestingly, no similar pathways could be found in the free state. The process targeting on the allosteric pathways can better regulate the glycolysis of PKM2 and significantly inhibit the progression of tumor.
| S-EPMC5115163 | biostudies-literature
Project description:In the study of allosteric proteins, understanding which effector-protein interactions contribute to allosteric activation is important both for designing allosteric drugs and for understanding allosteric mechanisms. The antihyperglycemic target, human liver pyruvate kinase (hL-PYK), binds its allosteric activator, fructose 1,6-bisphosphate (Fru-1,6-BP), such that the 1'-phosphate interacts with side chains of Arg501 and Trp494 and the 6'-phosphate interacts with Thr444, Thr446, Ser449 (i.e., the 444-449 loop), and Ser531. Additionally, backbone atoms from the 527-533 loop interact with a sugar ring hydroxyl and the two effector phosphate moieties. An effector analogue series indicates that only one phosphate on the sugar is required for activation. However, singly phosphorylated sugars, including Fru-1-P and Fru-6-P, bind with a Kix in the range of 0.07-1 mM. The second phosphate of Fru-1,6-BP causes tight effector binding, because this native effector has a Kix of 0.061 ?M. Glucose 1,6-bisphosphate and ribulose 1,5-bisphosphate bind in the 0.07-1 mM range. The contrast with a higher Fru-1,6-BP binding indicates specificity for the fructose sugar conformation. Site-directed random mutagenesis at each residue that contacts bound Fru-1,6-BP showed that a negative charge introduced at position 531 mimics allosteric activation, even in the absence of Fru-1,6-BP. Collectively, analogue and mutagenesis studies are consistent with the 527-533 loop playing a key role in allosteric function. Deletion mutations that shortened the 527-533 loop were expected to prevent formation of hydrogen bonds between backbone atoms on the loop and Fru-1,6-BP. Indeed, Fru-1,6-BP did not activate these loop-shortened mutant proteins. Previous structural comparisons of M1-PYK and M2-PYK indicate that the 527-533 loop makes interactions across a subunit interface when an activator is not present. Mutating the hL-PYK subunit interface interactions among Trp527, Arg528, and Asp499 mimics allosteric activation. Considered with published structures, these results are consistent with (1) the two phosphates of Fru-1,6-BP docking to Arg501/Trp494 and the 444-449 loop, respectively, and (2) the formation of hydrogen bonds among Fru-1,6-BP and backbone atoms of the 527-533 loop pulling this loop away from the subunit interface, which results in breaking of the Trp527-Arg528-Asp499 interactions to elicit an allosteric response.
| S-EPMC5286843 | biostudies-literature
Project description:The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37 degrees C. As a function of increasing pH, hL-PYK's affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and alanine slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to alanine is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for alanine inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response.
| S-EPMC2687410 | biostudies-literature
Project description:The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the concentration of the effector fructose 1,6-bisphosphate. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that an increase in the fructose bisphosphate concentration from 24 microM to 1.2 mM brings about a transition from a sigmoidal to a non-inflected form in the relationships v = f([phosphoenolpyruvate]) and v = f([Mg2+]) together with a large increase in the affinity of these substrates for the enzyme. The binding behaviour of ADP is barely affected by the same change in effector concentration. By contrast, increase in fructose bisphosphate concentration below 24 microM increases the affinity of the enzyme for all its substrates and the sigmoidicity of the corresponding velocity-substrate-concentration relationships. As a result of this change in behaviour it has been found impossible to represent all the data by the exponential model for a regulatory enzyme, and it is suggested (supported by comparisons with previous work) that the failure may reflect a secondary action of the effector upon the enzyme.
| S-EPMC1146627 | biostudies-other
Project description:Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is characterized by elevated glycogen levels and fat deposition. These consistent metabolic alterations are associated with normoxic stabilization of hypoxia-inducible factors (HIFs) secondary to von Hippel-Lindau (VHL) mutations that occur in over 90% of ccRCC tumours. However, kidney-specific VHL deletion in mice fails to elicit ccRCC-specific metabolic phenotypes and tumour formation, suggesting that additional mechanisms are essential. Recent large-scale sequencing analyses revealed the loss of several chromatin remodelling enzymes in a subset of ccRCC (these included polybromo-1, SET domain containing 2 and BRCA1-associated protein-1, among others), indicating that epigenetic perturbations are probably important contributors to the natural history of this disease. Here we used an integrative approach comprising pan-metabolomic profiling and metabolic gene set analysis and determined that the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) is uniformly depleted in over six hundred ccRCC tumours examined. Notably, the human FBP1 locus resides on chromosome 9q22, the loss of which is associated with poor prognosis for ccRCC patients. Our data further indicate that FBP1 inhibits ccRCC progression through two distinct mechanisms. First, FBP1 antagonizes glycolytic flux in renal tubular epithelial cells, the presumptive ccRCC cell of origin, thereby inhibiting a potential Warburg effect. Second, in pVHL (the protein encoded by the VHL gene)-deficient ccRCC cells, FBP1 restrains cell proliferation, glycolysis and the pentose phosphate pathway in a catalytic-activity-independent manner, by inhibiting nuclear HIF function via direct interaction with the HIF inhibitory domain. This unique dual function of the FBP1 protein explains its ubiquitous loss in ccRCC, distinguishing FBP1 from previously identified tumour suppressors that are not consistently mutated in all tumours.
| S-EPMC4162811 | biostudies-literature