Tissue-specific distribution of pyruvate kinase isoforms improve the physiological plasticity of Northern krill, Meganyctiphanes norvegica

The Northern krill, Meganyctiphanes norvegica (Crustacea, Euphausiacea) is widely distributed in the northern and northeastern parts of the Atlantic Ocean where it faces rapid variations in water temperatures and food. We studied the physiological potential of krill to compensate for environmentally induced metabolic changes. Two isoforms of the glycolytic key enzyme pyruvate kinase (PKI and PKII, EC 2.7.1.40) were partly purified from M. norvegica by anion exchange chromatography. Specific activities and catalytic properties of each isoform were determined in whole body extracts as well as in selected organs and tissues of males and females. Both PK-isoenzymes differed slightly in their temperature profiles, their activation energy and their molecular weights. PKI showed a high affinity for the substrate PEP and was not affected by fructose-1.6-bisphosphate (FBP). In contrast, PKII showed low affinity for PEP but was strongly activated by FBP, up to 40-fold. The specific PK-activity of whole organisms was lower in females (44.9 ± 4.8 U·g_ww^− 1) than in males (61.3 ± 7.7 U·g_ww^− 1). In females PK II represented 20% of the total PK-activity while it was only 10% in males. Highest PK activities were present in the hearts, the eyes, pleopods and in the thoracopods. In the stomachs and the midgut glands PK activities were low. Almost all organs contained PKI and PKII. However, PKI prevailed in the abdomens, the pleopods, the thoracopods, and in the thoracic muscles. PKII dominated in the eyes, the midgut glands and in the ovaries. Experiments showed that the tissue concentrations of FBP increased with food uptake and temperature. The expression of two PK-isoforms with different kinetic properties and the mediation of substrate affinity by FPB is a powerful tool to immediately regulate glycolytic energy flows in different organs. The krill is capable of adjusting energy consumption to changes in nutritional conditions as well as variations of environmental temperatures.     

Kinetic and allosteric consequences of mutations in the subunit and domain interfaces and the allosteric site of yeast pyruvate kinase.

The mechanism by which pyruvate kinase (PK) is allosterically activated by fructose-1,6-bisphosphate (FBP) is poorly understood. To identify residues key to allostery of yeast PK, a point mutation strategy was used. T403E and R459Q mutations in the FBP binding site caused reduced FBP affinity. Introducing positive charges at the 403, 458, and 406 positions in the FBP binding site had little consequence. The mutation Q299N in the A [bond] A subunit interface caused the enzyme response to ADP to be sensitive to FBP. The T311M A [bond] A interface mutant has a decreased affinity for PEP and FBP, and is dependent on FBP for activity. The R369A mutation in the C [bond] C interface only moderately influenced allostery. Creating an E392A mutation in the C [bond] C subunit interface eliminated all cooperativity and allosteric regulation. None of the seven A [bond] C domain interface mutations altered allostery. A model that includes a central role for E392 in allosteric regulation of yeast PK is proposed.     

Kinetic and expression profiling of cytosolic pyruvate kinase enzyme during seed development of Indian mustard (Brassica juncea)

In the recent years tremendous progress has been achieved in deconstructing the oil biosynthetic pathways, majority of which is in Arabidopsis. Glycolysis is fundamental to this process as it is the cardinal supplier of precursors for fatty acid metabolism. Recent reports suggest that modification and expression of pyruvate kinase (PK), a crucial regulatory enzyme involved in glycolysis is one of the plausible ways to alter seed oil content in plants. In the present study we evaluated the kinetic behavior and expression profiling of pyruvate kinase, associated with seed development in a major oilseed crop B. juncea. Developmental profiling of the enzyme showed that enzyme activity was highest during middle stage (35 DAF) of seed development which is strongly corroborated by the expression profiling of the enzyme using RT-PCR approach. Oil accumulation pattern also correlated with the enzyme expression study. Comparative activity profiling from different tissues showed seedlings to have elevated activity than other tissues. For kinetic characterization, the enzyme was partially purified by 12.3 fold using DEAE-Sephadex column and showed a narrow pH optimum of 7.0. In presence of saturated substrate concentration, the enzyme exhibited hyperbolic kinetics for both ADP and PEP with Km (Michaelis constant value) for PEP and ADP was found to be 178.5 and 96.45 μM respectively. ATP and citrate are the most significant allosteric effectors of the partially purified PK. Study on isozymes of PK resulted in a single band.     

Identification of novel allosteric regulators of human-erythrocyte pyruvate kinase

Erythrocyte pyruvate kinase (PK) is an important glycolytic enzyme, and manipulation of its regulatory behavior by allosteric modifiers is of interest for medicinal purposes. Human-erythrocyte PK was expressed in Rosetta cells and purified on an Ni-NTA column. A search of the small-molecules database of the National Cancer Institute (NCI), using the UNITY software, led to the identification of several compounds with similar pharmacophores as fructose-1,6-bisphosphate (FBP), the natural allosteric activator of the human kinases. The compounds were subsequently docked into the FBP binding site using the programs FlexX and GOLD, and their interactions with the protein were analyzed with the energy-scoring function of HINT. Seven promising candidates, compounds 1-7, were obtained from the NCI, and subjected to kinetics analysis, which revealed both activators and inhibitors of the R-isozyme of PK (R-PK). The allosteric effectors discovered in this study could prove to be lead compounds for developing medications for the treatment of hemolytic anemia, sickle-cell anemia, hypoxia-related diseases, and other disorders arising from erythrocyte PK malfunction.     

Crystal structure of pyruvate kinase from Geobacillus stearothermophilus

The pyruvate kinase (PK) from a moderate thermophile, Geobacillus stearothermophilus, is an allosteric enzyme activated by AMP and ribose 5-phosphate but not fructose 1, 6-bisphosphate (FBP), which is a common activator of PKs. It has an extra C-terminal sequence (ECTS), which contains a highly conserved phosphoenolpyruvate (PEP) binding motif, but its function and structure remain unclear. To elucidate the structural characteristics of the effector-binding site and the ECTS, the crystal structure of the C9S/C268S mutant of the enzyme was determined at 2.4 A resolution. The crystal belonged to space group P6(2)22, with unit cell parameters a, b = 145.97 A, c = 118.03 A. The enzyme was a homotetramer and its overall domain structure was similar to the previously solved structures except that the ECTS formed a new domain (C' domain). The structure of the C' domain closely resembled that of the PEP binding domain of maize pyruvate phosphate dikinase. A sulphate ion was found in a pocket in the effector-binding C domain. This site corresponds to the 6-phosphate group-binding site in yeast PK bound FBP and seems to be the effector-binding site. Through comparison of the structure of the putative effector-binding site to that of the FBP binding site of the yeast enzyme, the structural basis of the effector specificity of the G. stearothermophilus PK is discussed.     

The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase

For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S_0.5 value 10³-fold and increased the V_max value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus l-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172–185) and MR2 (positions 211–221), form a compact core for allosteric motion, and His¹⁷⁹ of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased V_max 4-fold but reduced pyruvate S_0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase V_max, but 10²-reduced pyruvate S_0.5, and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule.     

Phosphoenolpyruvate and Mg Binding to Pyruvate Kinase Monitored by Infrared Spectroscopy

Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg²⁺ binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg²⁺ binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-¹³C₂-labeled PEP. We obtained the following assignments: 1589 cm⁻¹ (antisymmetric carboxylate stretching vibration); 1415 cm⁻¹ (symmetric carboxylate stretching vibration); 1214 cm⁻¹ (C-O stretching vibration); 1124 and 1110 cm⁻¹ (asymmetric PO₃^2- stretching vibrations); and 967 cm⁻¹ (symmetric PO₃^2- stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm⁻¹. The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na⁺ exhibited the same band positions as in the presence of K⁺, indicating very similar interaction strengths between ligand and protein in both cases.     

Resiniferonol-related Diterpene Esters from Daphne odora Thunb. and their Ornithine Decarboxylase-inducing Activity in Mouse Skin

Phosphoenolpyruvate (PEP) carboxylase purified from Brevibacterium flavum was specifically activated by fructose 1,6-bisphosphate (FBP). The other intermediates of sugar metabolism or their structural analogues did not influence the activity. FBP decreased the apparent Km for PEP but did not affect that for another substrate, bicarbonate, or the apparent maximum velocity for PEP. The dissociation constants for FBP from enzyme-FBP and enzyme-PEP-FBP complex were 63 and 32 μm, respectively, being almost equivalent to those for acetyl-CoA. Synergistic activation by FBP and acetyl-CoA was not observed with the B. flavum enzyme, unlike the Escherichia coli enzyme. FBP, like acetyl-CoA, was kinetically competitive with aspartate. With respect to another feedback inhibitor, 2-oxoglutarate, acetyl-CoA was non-competitive, whereas FBP was of mixed-type, i.e., FBP but not acetyl-CoA prevented 2-oxoglutarate from binding to the enzyme to a certain extent. Homotropic cooperativity was observed only with FBP but not with acetyl-CoA in the absence of inhibitors. Cooperativities of FBP and acetyl-CoA were increased by aspartate but not by 2-oxoglutarate. In the aspartate-overproducing mutant enzyme, the Michaelis constant for PEP was decreased, whereas the inhibitor constant for aspartate with or without simultaneous addition of 2-oxoglutarate and the activator constants for FBP and acetyl-CoA were increased. The decreased Michaelis constant for PEP was comparable to the apparent Km of the wild-type enzyme for PEP in the presence of the saturated concentration of FBP, and would result in a further decrease in the affinity of the mutant enzyme for aspartate.     

Changes in Sensitivity to Effectors of Maize Leaf Phosphoenolypyruvate Carboxylase during Light/Dark Transitions

Illumination of previously darkened maize (Zea mays L. cv Golden Cross Bantam T51) leaves had no effect on the concentration of phosphoenolpyruvate (PEP) carboxylase protein, but increased enzyme activity about 2-fold when assayed under suboptimal conditions (pH 7.0 and limiting PEP). In addition, sensitivity to effectors of PEP carboxylase activity was significantly altered; e.g. malate inhibition was reduced and glucose-6-phosphate activation was increased. Consequently, 10- to 20-fold differences in PEP carboxylase activity were observed during dark to light transitions when assayed in the presence of effectors. At pH 7.0 activity of purified PEP carboxylase was not proportional to enzyme concentrations. Below 0.7 microgram PEP carboxylase protein per milliliter, enzyme activity was disproportionately reduced. Including polyethylene glycol plus potassium chloride in the reaction mixture eliminated this discontinuity and substantially increased PEP carboxylase activity and reduced malate inhibition dramatically. Inclusion of polyethylene glycol in the assay mixture specifically increased the activity of PEP carboxylase extracted from dark leaves, and reduced malate inhibition of the enzyme from both light and dark leaves. Collectively, the results suggest that PEP carboxylase in maize leaves is subjected to some type of protein modification that affects both activity and effector sensitivity. We postulate that changes in quaternary structure (dissociation or altered subunit interactions) may be involved.     

Lactate Contributes to Glyceroneogenesis and Glyconeogenesis in Skeletal Muscle by Reversal of Pyruvate Kinase

Phosphoenolpyruvate (PEP) generated from pyruvate is required for de novo synthesis of glycerol and glycogen in skeletal muscle. One possible pathway involves synthesis of PEP from the citric acid cycle intermediates via PEP carboxykinase, whereas another could involve reversal of pyruvate kinase (PK). Earlier studies have reported that reverse flux through PK can contribute carbon precursors for glycogen synthesis in muscle, but the physiological importance of this pathway remains uncertain especially in the setting of high plasma glucose. In addition, although PEP is a common intermediate for both glyconeogenesis and glyceroneogenesis, the importance of reverse PK in de novo glycerol synthesis has not been examined. Here we studied the contribution of reverse PK to synthesis of glycogen and the glycerol moiety of acylglycerols in skeletal muscle of animals with high plasma glucose. Rats received a single intraperitoneal bolus of glucose, glycerol, and lactate under a fed or fasted state. Only one of the three substrates was ¹³C-labeled in each experiment. After 3 h of normal awake activity, the animals were sacrificed, and the contribution from each substrate to glycogen and the glycerol moiety of acylglycerols was evaluated. The fraction of ¹³C labeling in glycogen and the glycerol moiety exceeded the possible contribution from either plasma glucose or muscle oxaloacetate. The reverse PK served as a common route for both glyconeogenesis and glyceroneogenesis in the skeletal muscle of rats with high plasma glucose. The activity of pyruvate carboxylase was low in muscle, and no PEP carboxykinase activity was detected.     

Steady-state fluorescence of Escherichia coli phosphofructokinase reveals a regulatory role for ATP.

We have investigated the effects of ligands and effectors on the intrinsic fluorescence of Escherichia coli phosphofructokinase (PFK). We have found that the substrate fructose 6-phosphate (Fru6P) or the allosteric activator ADP can quench the fluorescence up to 35%. The response is hyperbolic with Ks[Fru6P] of 20 microM and Ks[ADP] of 13 microM. The allosteric inhibitor phosphoenolpyruvate (PEP) converts the hyperbolic response with respect to Fru6P to a sigmoidal response. AMP-PNP, a nonhydrolyzable analogue of ATP, also inhibits the Fru6P fluorescence response. PFK mutant KA213, which is insensitive to effectors, has a decreased fluorescence response with respect to ADP, and PEP does not convert the Fru6P response to sigmoidicity. However, its fluorescence response with respect to Fru6P is decreased by ATP or AMP-PNP. Taken together, these results suggest that, in the absence of effectors or ligands, E. coli PFK exists in a state with high affinity for Fru6P ("R" state). This state can be altered to a low affinity ("T" state) by PEP binding to the allosteric site or by ATP binding to the enzyme.     

Light/dark modulation of phosphoenolpyruvate carboxylase in C3 and C4 species

In this report, the effects of light on the activity and allosteric properties of phosphoenolpyruvate (PEP) carboxylase were examined in newly matured leaves of several C₃ and C₄ species. Illumination of previously darkened leaves increased the enzyme activity 1.1 to 1.3 fold in C₃ species and 1.4 to 2.3 fold in C₄ species, when assayed under suboptimal conditions (pH 7) without allosteric effectors. The sensitivities of PEP carboxylase to the allosteric effectors malate and glucose-6-phosphate were markedly different between C₃ and C₄ species. In the presence of 5 mM malate, the activity of the enzyme extracted from illuminated leaves was 3 to 10 fold higher than that from darkened leaves in C₄ species due to reduced malate inhibition of the enzyme from illuminated leaves, whereas it increased only slightly in C₃ species. The Ki(malate) for the enzyme increased about 3 fold by illumination in C₄ species, but increased only slightly in C₃ species. Also, the addition of the positive effector glucose-6-phosphate provided much greater protection against malate inhibition of the enzyme from C₄ species than C₃ species. Feeding nitrate to excised leaves of nitrogen deficient plants enhanced the degree of light activation of PEP carboxylase in the C₄ species maize, but had little or no effect in the C₃ species wheat. These results suggest that post-translational modification by light affects the activity and allosteric properties of PEP carboxylase to a much greater extend in C₄ than in C₃ species.     

Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase.

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.     

Kinetic properties of pyruvate kinase isolated from rat hepatic tumours.

The results demonstrate the existence of L and M forms of pyruvate kinase in rat hepatomas. Tumours were induced by feeding N-Nitrosodiethylamine. The kinetic properties of the L-type tumour enzyme was markedly different from the L-enzyme form found in normal liver. The L-form of tumour enzyme was purified by DEAE cellulose-Sephadex G200 chromatography (Sp. activity 41 units/mg). $\text{MgADP}{}^{\mathrm{?}}\text{ADP}{}^{\mathrm{2?}}$ of $\text{20}\text{1}$ gave optimum activity for both the intrinsic and F1,6di-P stimulated reactions. ATP did not inhibit the enzyme. Alanine (2.5 nM) caused 60% inhibition at low PEP concentrations (0.25 mM). The homotropic effector (PEP) exhibited a complex allosteric pattern and saturation kinetics were not observed for either the intrinsic or F1,6di-P stimulated reactions with PEP concentrations as high as 10 mM.     

Regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase activity in adult Fasciola hepatica (Trematoda).

F. hepatica pyruvate kinase and phosphoenolpyruvate (PEP) carboxykinase were found to have properties of regulatory enzymes in the dissimilation of PEP and the control of metabolic flow. Mn²⁺ and K⁺ were required for pyruvate kinase activity. In the presence of fructose-1, 6-diphosphate (FDP), Mg²⁺ could substitute for Mn²⁺. FDP caused a 4-fold increase in the Mn²⁺ activated pyruvate kinase activity. This was accompanied by a 12-fold decrease in apparent K_m(PEP) and a 3-fold decrease in apparent K_{m (ADP)}. ATP markedly inhibited F. hepatica pyruvate kinase, but this inhibition was relieved by FDP. Estimates of metabolic levels indicated that the pyruvate kinase is saturated with PEP and ADP in vivo, but will be highly sensitive to fluctuations in the physiological concentrations of FDP and ATP. NADH doubled the activity of the PEP carboxykinase reaction and decreased the apparent K_{m (PEP)} for this enzyme 3-fold. While the maximal activity of the PEP carboxykinase reaction was substantially higher than the pyruvate kinase reaction, the steady state concentration of PEP suggests that the PEP carboxykinase will not be saturated with this substrate.     

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO₂. They are activated by Mn²⁺, a metal ion that coordinates to the protein through the ?-amino group of a lysine residue, the N_?-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ?-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ?-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn²⁺ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn²⁺ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn²⁺. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

Determinants of allosteric activation of yeast pyruvate kinase and identification of novel effectors using computational screening

We have analyzed the structural determinants of the allosteric activation of yeast pyruvate kinase (YPK) by mutational and kinetic analysis and initiated a structure-based design project to identify novel effectors that modulate its allosteric response by binding to the allosteric site for fructose-1,6-bisphosphate (FBP). The wild-type enzyme is strongly activated by fructose-1,6-bisphosphate and weakly activated by both fructose-1-phosphate and fructose-6-phosphate; the strength of the activation response is proportional to the affinity of the allosteric effector. A point mutation within the 6'-phosphate binding loop of the allosteric site (T403E) abolishes activation of the enzyme by fructose-1, 6-bisphosphate. The mutant enzyme is also not activated by F1P or F6P. The mutation alone (which incorporates a glutamic acid that is strictly conserved in mammalian M1 isozymes) slightly reduces cooperativity of substrate binding. Three novel compounds were identified that effect the allosteric regulation of YPK by FBP and/or act as novel allosteric activators of the enzyme. One is a physiologically important diphospho sugar, while the other two are hydrophobic compounds that are dissimilar to the natural effector. These results demonstrate that novel allosteric effectors may be identified using structure-based screening and are indicative of the potential of this strategy for drug discovery. Regulatory sites are generally more divergent than catalytic sites and therefore offer excellent opportunities for discrimination and specificity between different organisms or between different tissue types.     

Pyruvate kinase in pig liver mitochondria

The existence of the pyruvate kinase (PK) in pig liver mitochondria was shown by monitoring photometrically the PK reaction in solubilised mitochondria with either phosphoenolpyruvate (PEP) or ADP used as a substrate. In distinction with the cytosolic isoenzyme, the mitochondrial PK showed a sigmoidal dependence on either PEP or ADP concentrations. The occurrence of the mitochondrial PK was confirmed by immunological analysis. Titration with digitonin showed that mPK is restricted to the matrix. PEP addition to mitochondria resulted in reduction of the intramitochondrial NAD(P)⁺ inhibited by either the non-penetrant thiol reagent mersalyl or by arsenite, an inhibitor of the pyruvate dehydrogenase complex. Citrate/oxaloacetate appearance outside mitochondria also occurred as result of PEP addition to PLM. Taken together these findings support a role for PEP itself in triggering fatty acid synthesis via its mitochondrial metabolism.     

From genome to enzyme: analysis of key glycolytic and oxidative pentose-phosphate pathway enzymes in the cyanobacterium Synechocystis sp. PCC 6803

Activities of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphofructokinase (PFK), enolase, pyruvate kinase (PK) and phosphoenolpyruvate (PEP) carboxylase were determined in extracts of photoautotrophic, mixotrophic, and heterotrophic cultures of Synechocystis sp. PCC 6803. Annotated genomes of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 were analyzed for the respective predicted physical properties of each enzyme investigated here. Enzymatic activity was largely unaffected by nutritional mode, with the exception of glucokinase and PK whose activities were significantly elevated in heterotrophic cultures of Synechocystis sp. PCC 6803. PFK activity was insensitive to bacterial PFK-A (allosteric) effectors such as PEP, implying that Synechocystis PFK should be classified as a PFK-B (non-allosteric). Immunoblot and kinetic studies indicated that irrespective of nutritional mode, the Synechocystis PK corresponds to a PK-A (AMP activated) rather than PK-F (fructose-1,6-bisphosphate activated).