Dimethylsulfoxide promotes K+-independent activity of pyruvate kinase and the acquisition of the active catalytic conformation.

Pyruvate kinase requires K+ for maximal activity; the enzyme exhibits 0.02% of maximal activity in its absence [Kayne, F. J. (1971) Arch. Biochem. Biophys. 143, 232-239]. However, pyruvate kinase entrapped in reverse micelles exhibits an important K+-independent activity [Ramírez-Silva, L., Tuena de Gómez-Puyou, M., & Gómez-Puyou, A. (1993) Biochemistry 32, 5332-5338]. It is possible that the amount of water, as well as interactions of the protein with the micelles, can account for this behavior. We therefore explored the solvent effects on the catalytic properties of muscle pyruvate kinase. The enzyme exhibited an activity of 19.4 micromol x min(-1) x mg(-1) in 40% dimethylsulfoxide, compared with 280 and 0.023 micromol x min(1) x mg(-1) observed with and without K+ in water, respectively. pH activity profiles and kinetic constants for the substrates of pyruvate kinase in dimethylsulfoxide without K+ were similar to those in 100% water with K+, and differed from those in water without K+. The spectral center of mass of the emission spectrum of pyruvate kinase in 100% water exhibited a blue shift of 3.5 nm in the presence of Mg(2+), phosphenolpyruvate, and K+, ligands that induce the active conformation of the enzyme. The spectral center of mass of the apoenzyme in 30-40% dimethylsulfoxide coincided with that of the enzyme-Mg(2+)-phosphenolpyruvate-K+ complex in 100% water. The water relaxation rate enhancement factor and binding of phosphenolpyruvate to the pyruvate kinase-Mn(2+)-(CH3)4N+ complex in 30-40% dimethylsulfoxide were similar to those of the pyruvate kinase-Mn(2+)-K+ complex in water. The aforementioned results indicate that when muscle pyruvate kinase is without K+, 30-40% dimethylsulfoxide induces its active conformation.     

L-Phenylalanine inhibition of muscle pyruvate kinase

The allosteric inhibition of M_l-type pyruvate kinase from rabbit skeletal muscle by phenylalanine is reciprocally dependent on Mg²⁺ and phosphoenolpyruvate concentrations . At pH 8, phenylalanine acts as a competitive inhibitor with respect to Mg²⁺ and phosphoenolpyruvate, and vice versa. Phenylalanine introduces sigmoidicity into the dependence of the reaction velocity on [Mg²⁺]. In vitro kinetic analysis indicates that phenylalanine inhibition of muscle pyruvate kinase is unlikely to have regulatory significance in vivo.     

Vanadyl(IV) complexes with pyruvate kinase: activation of the enzyme and electron paramagnetic resonance properties of ternary complexes with the protein

Complexes of the oxocation of vanadyl(IV), VO2+, with pyruvate kinase from rabbit muscle have been investigated by steady-state kinetic assays and by EPR spectroscopy. Pyruvate kinase requires 2 eq of divalent cation for activity. VO2+ alone is a poor activator of the normal physiological reaction catalyzed by the enzyme and of the enzyme-catalyzed exchange of the methyl protons of pyruvate with solvent. VO2+ alone is, however, an activator of the enzyme-catalyzed phosphorylation of glycolate by ATP. VO2+ is more effective than Mg2+ in activation of the bicarbonate-dependent ATPase reaction of pyruvate kinase, and in the enzyme-catalyzed hydrolysis of phosphoenolpyruvate. EPR data show that VO2+ binds to the divalent cation site on the protein competitively with respect to Mg2+. The VO2+-enzyme complex has a high affinity for bicarbonate. Direct coordination of pyruvate, oxalate, and glycolate to the enzyme-bound VO2+ has been established by EPR measurements with specifically 17O-labeled forms of these compounds.     

Pyruvate kinase from Chlamydia trachomatis is activated by fructose-2,6-bisphosphate

Pyruvate kinase is the final regulatory point in the catabolic Embden-Meyerhoff-Parnas pathway, which controls the carbon flux of glycolytic intermediates and regulates the level of ATP in the cell. In a previous study, we identified, cloned and sequenced pyruvate kinase from the obligate intracellular bacterium Chlamydia trachomatis and demonstrated that the enzyme was active in crude extract. Here, we report the kinetic properties of highly purified C. trachomatis pyruvate kinase. The results indicate that C. trachomatis pyruvate kinase is 53.5 kDa with a pH optima of 7.3. Kinetic studies show that C. trachomatis pyruvate kinase requires both K+ and Mg2+ ions for activity, exhibits sigmoidal kinetics with respect to phosphoenolpyruvate and Michaelis-Menten kinetics with respect to ADP. In addition, C. trachomatis pyruvate kinase is able to use alternative nucleoside diphosphates as phosphate acceptors, although it shows the greatest activity with ADP. In contrast to other bacterial pyruvate kinases that are activated by AMP, our data show that AMP, in addition to ATP and GTP, inhibits C. trachomatis pyruvate kinase. Surprisingly, unlike any other known bacterial pyruvate kinase, C. trachomatis pyruvate kinase was allosterically activated by fructose-2,6-bisphosphate, an important regulatory metabolite that has only been reported in eukaryotes.     

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.     

Regulation of mammalian pyruvate dehydrogenase

In mammalian tissues, two types of regulation of the pyruvate dehydrogenase complex have been described: end product inhibition by acetyl CoA and NADH: and the interconversion of an inactive phosphorylated form and an active nonphosphorylated form by an ATP requiring kinase and a specific phosphatase. This article is largely concerned with the latter type of regulation of the complex in adipose tissue by insulin (and other hormones) and in heart muscle by lipid fuels. Effectors of the two interconverting enzymes include pyruvate and ADP which inhibit the kinase, acetoin which activates the kinase and Ca2+ and Mg2+ which both activate the phosphatase and inhibit the kinase. Evidence is presented that all components of the pyruvate dehydrogenase complex including the phosphatase and kinase are located within the inner mitochondrial membrane. Direct measurements of the matrix concentration of substrates and effectors is not possible by techniques presently available. This is the key problem in the identification of the mechansims involved in the alterations in pyruvate dehydrogenase activity observed in adipose tissue and muscle. A number of indirect approaches have been used and these are reviewed. Most hopeful is the recent finding in this laboratory that in both adipose tissue and heart muscle, differences in activity of pyruvate dehydrogenase in the intact tissue persist during preparation and subsequent incubation of mitochondria.     

Pancreatic islets contain the M2 isoenzyme of pyruvate kinase its phosphorylation has no effect on enzyme activity

To determine which of the major isoenzymes of pyruvate kinase pancreatic islet pyruvate kinase most resembled, it was compared to pyruvate kinase from other tissues in kinetic and immunologic studies. The pattern of activation by fructose bisphosphate and the patterns of inhibition by alanine and phenylalanine were most similar to those of the M₂ isoenzyme from kidney and were dissimilar to those of the isoenzymes from skeletal muscle (type M₁) and liver (type L). The islet pyruvate kinase was inhibited by anti-M₁ pyruvate kinase serum (which crossreacts with the M₂ isoenzyme), but not by anti-L pyruvate kinase. These results are most consistent with islets possessing predominantly, if not exclusively, the M₂ isoenzyme of pyruvate kinase. We previously showed that rat pancreatic islet cytosol contains protein kinases that can catalyze a calcium-activated phosphorylation of an endogenous peptide that has properties, such as subunit molecular weight and isoelectric pH, that are identical to those of the M₂ and M, isoenzymes of pyruvate kinase, and that islet cytosol can catalyze phosphorylation of muscle pyruvate kinase. In the present study it was shown that incubating islet cytosol with ATP under conditions known to permit phosphorylation and inhibition of liver pyruvate kinase did not affect the islet pyruvate kinase activity. It is concluded that phosphorylation of the islet pyruvate kinase has no immediate effect on enzyme activity.Abbreviations EGTA ethylene glycos his (-aminoethyl ether)-N,N,NN-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

A kinetic study of the pH effect on the allosteric properties of pyruvate kinase from Phycomyces blakesleeanus

This paper reports the pH-dependence of the allosteric kinetics of Phycomyces blakeseeanus pyruvate kinase with phosphoenol pyruvate and Mg2+ ions in the presence and in the absence of fructose 1,6-bisphosphate (allosteric activator) and L-alanine (allosteric inhibitor). Hydrogen ions increase the affinity of the inhibitory binding sites for phosphoenol pyruvate and Mg2+ ions. Assuming partial conformational states of high and low affinity for inhibitory binding sites, the data presented are in good agreement with the predictions postulated by the two-state concerted-symmetry model of Monod, Wyman, and Changeux. Fructose-1,6-bisphosphate and L-alanine show opposite effects on the interactions of phosphoenol pyruvate and Mg2+ ions with their respective catalytic and inhibitory binding sites. At pH 6.0, the regulation of the Phycomyces pyruvate kinase activity by the concentrations of phosphoenol pyruvate and Mg2+ ions is controlled mainly by L-alanine.     

Affinity labeling of rabbit muscle pyruvate kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Rabbit muscle pyruvate kinase is irreversibly inactivated upon incubation with the adenine nucleotide analogue, 5'-p-fluorosulfonylbenzoyladenosine. A plot of the time dependence of the logarithm of the enzymatic activity at a given time divided by the initial enzymatic activity(logE/Eo) reveals a biphasic rate of inactivation, which is consistent with a rapid reaction to form partially active enzyme having 54% of the original activity, followed by a slower reaction to yield totally inert enzyme. In addition to the pyruvate kinase activity of the enzyme, modification with 5'-p-fluorosulfonylbenzoyladenosine also disrupts its ability to catalyze the decarboxylation of oxaloacetate and the ATP-dependent enolization of pyruvate. In correspondence with the time dependence of inactivation, the rate of incorporation of 5'-p-[14C]fluorosulfonylbenzoyladenosine is also biphasic. Two moles of reagent per mole of enzyme subunit are bound when the enzyme is completely inactive. The pseudo-first-order rate constant for the rapid rate is linearly dependent on reagent concentration, whereas the constant for the slow rate exhibits saturation kinetics, suggesting that the reagent binds reversibly to the second site prior to modification. The adenosine moiety is essential for the effectiveness of 5'-p-fluorosulfonylbenzoyladenosine, since p-fluorosulfonylbenzoic acid does not inactivate pyruvate kinase at a significant rate. Thus, the reaction of 5'-p-fluorosulfonylbenzoyladenosine with pyruvate kinase exhibits several of the characteristics of affinity labeling of the enzyme. Protection against inactivation by 5'-p-fluorosulfonylbenzoyladenosine is provided by the addition to the incubation mixture of phosphoenolpyruvate. Mg-ADP or Mg2+. In contrast, the addition of pyruvate, Mg-ATP, or ADP and ATP alone has no effect on the rate of inactivation. These observations are consistent with the postulate that the 5'-p-fluorosulfonylbenzoyladenosine specifically labels amino acid residues in the binding region of Mg2+ and the phosphoryl group of phosphoenolpyruvate which is transferred during the catalytic reaction. The rate of inactivation increases with increasing pH, and k1 depends on the unprotonated form of an amino acid residue with pK = 8.5. On the basis of the pH dependence of the reaction of pyruvate kinase with 5'-p-fluorosulfonylbenzoyladenosine and the elimination of cysteine residues as possible sites of reaction, it is postulated that lysyl or tyrosyl residues are the most probably candidates for the critical amino acids.     

Purification and properties of rat brain pyruvate kinase

Rat brain pyruvate kinase was purified to near homogeneity by a three-step process involving ammonium sulfate precipitation and phosphocellulose and Blue-Sepharose CL-6B column chromatography. The enzyme migrated on polyacrylamide gel along with a commercial sample of rabbit muscle pyruvate kinase. The enzyme showed a hyperbolic relationship with phosphoenolpyruvate and ADP, with apparent Km's of 0.18 and 0.42 X 10(-3) M, respectively. The enzyme was inhibited by ATP, the effect being more pronounced at unsaturating concentrations of phosphoenolpyruvate. L-Phenylalanine was found to be a strong inhibitor of the enzyme, with the Ki for inhibitor being 0.11 mM. The inhibition by phenylalanine was more pronounced at pH 7.4 than at pH 7.0, and appeared to be competitive with phosphoenolpyruvate. L-Alanine and fructose 1,6-bisphosphate prevented the inhibition of the enzyme by phenylalanine. Ca2+ was found to be a strong inhibitor of the enzyme, and the inhibition was more marked at saturating phosphoenolpyruvate concentrations. The kinetic properties of the purified brain pyruvate kinase suggest that the enzyme may be distinct from the muscle or liver enzymes.     

Plant Pyruvate Kinase

Pyruvate kinase is an important enzyme of glycolytic pathway that also functions in providing carbon skeleton for fatty acid biosynthesis. It has been purified to near homogeneity from Ricinus communis, Selenastrum minutum, Cynodon dactylon, Brassica campestris and B. napus, and characterised. Partially purified preparations are reported from several other sources. A phosphoenolpyruvate (PEP) phosphatase accompanies pyruvate kinase. In plants, two isozymes of pyruvate kinase are reported, namely cytosolic and plastidic. Isoforms of cytosolic pyruvate kinase have also been reported from spinach. In most cases pyruvate kinase is a tetrameric protein and the molecular mass lies between 200 to 250 kDa. The pH optimum is in the range of 6.2 to 7.5. It requires both Mg²⁺ and K⁺ for maximum activity. ATP, citrate, and oxalate inhibit pyruvate kinase in most cases. A sequential compulsory ordered mechanism of binding of substrates to the enzyme has been proposed.     

Effects of estradiol and testosterone on the activity of pyruvate kinase of the cardiac and skeletal muscles of rats as a function of age and sex

The specific activities of pyruvate kinase of cardiac and skeletal (gastrocnemius) muscles of adult rats of both sexes are lower than those of immature rats. The activity does not change after adulthood in the cardiac muscle, but decreases in the gastrocnemius. The activity of pyruvate kinase of the heart of immature and adult rats of both sexes decreases after castration, but is unaffected in old rats. Castration has no effect on the activity of pyrovate kinase of the gastrocnemius muscle of rats of both sexes at any age. In invo administration of estradiol (50 μg/100 g body weight) increases the activity of pyruvate kinase of the heart of castrated male and female rats of the three ages. For the skeletal muscle, the activity increases in castrated adult female and old male rats only. A higher dose (100 μg) of estradiol has variable effects on pyruvate kinase of the heart of male and female castrated rats of different ages. This dose increase pyruvate kinase significantly in the skeletal muscle of old castrated male and female rats. However, it decreases it in the skeletal muscle of adult castrated male rats. Testosterone (100 μm) increases the activity of pyruvate kinase of the heart of castrated male rats. This increase is lower in old age. It has no effect in the heart of castrated female rats of any age. Testosterone (50 μg) increases pyruvate kinase activity of the skeletal muscle of young ovariectomized rats only. A higher dose (100 μg) causes a significant increase in pyruvate kinase of the skeletal muscle of castrated adult and old male, and young and adult female rats, respectively. These data show that sex steroid hormones induce pyruvate kinase of striated muscles, and that the age- and sex-dependent variations may be due to changes in the levels of receptor proteins.     

Comparison of glycogen phosphorylase kinases of various rat tissues

Glycogen phosphorylase kinases in soluble fractions of various rat tissues were examined for the pH 6.8/8.5 activity ratio, Ca2+-dependency, activation by cyclic AMP-dependent protein kinase (protein kinase A), and reactivity with anti-skeletal muscle phosphorylase kinase serum. The enzymes could be divided into at least two major groups; muscle and liver types. The muscle type, that has a low value of pH 6.8/8.5 activity ratio, is highly dependent on Ca2+, markedly activated by protein kinase A, and strongly inhibited by the antiserum. Inversely, the liver type, that has a high value of pH 6.8/8.5 activity ratio, is poorly dependent on Ca2+, not activated by protein kinase A, and weakly inhibited by the antiserum. The enzymes from heart and skeletal muscle were similar and belonged to the former entity. Whereas, the enzymes from liver, kidney, spleen, lung, and testis appeared to belong to the latter entity. The enzyme from brain apparently differs from these entities, and seems to be an intermediate type or a hybrid of the two.     

Properties of pyruvate kinase and flagellar ATPase in rabbit spermatozoa: relation to metabolic strategy of the sperm cell

Rabbit sperm pyruvate kinase remains bound to the cell structure of hypotonically treated mature rabbit epididymal spermatozoa (HTRES). It displays kinetic behavior very similar to that of rabbit muscle pyruvate kinase with regard to KM values for substrates, activation by monovalent and divalent cations, inhibition by phenylalanine which is reversed by alanine, and lack of activation by fructose-1,6-biphosphate. The flagellar ATPase also remains bound to the cell structure of HTRES, whose motility may be reactivated by a source of ATP. It requires Mg+2 for activity; the KM for both ATP and MG+2 is 0.2 mM, implying that MgATP is the substrate. The ATPase activity is not inhibited by ouabain, oligomycin, or vanadate, which also do not affect reconstituted motility, and is not affected by cyclic AMP in the presence of an inhibitor of phosphodiesterase. The activities of pyruvate kinase and the flagellar ATPase in a given preparation of HTRES are comparable. Rabbit spermatozoa have a metabolic strategy which is very similar to muscle cells. This suggests that the major use of the sperm cell's metabolic machinery is maintenance of energy for the contractile work of motility and that only minor amounts of metabolic energy appear to be consumed in other reactions, including those involved in fertilization.     

Apparent inhibition of pyruvate kinase by phosphocreatine and phosphoarginine.

1. Addition of a non-dialysable, heat-labile and acid-precipitable factor which was not absorbed on DEAE-cellulose column, could restore the sensitivity of the chromatographed muscle pyruvate kinase from Marphysa sanguinea towards phosphocreatine inhibition. 2. This factor, being non-specific as it acts on pyruvate kinase isozymes from different sources, demonstrated high creatine kinase activity. 3. High concentrations of ADP, creatine or replacement of ADP with IDP/UDP or high pH abolished the inhibition indicating that the inhibition was mediated through creatine kinase by depleting ADP. 4. Apparent inhibition of phosphocreatine was related to the relative activities of 3 intracellular enzymes--pyruvate kinase, creatine kinase and adenosine triphosphatase.     

Secondary kinase reactions catalyzed by yeast pyruvate kinase.

1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. Wth Mg2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-biphosphate. With Mn2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on three secondary kinase reactions was also examined. 4. Results are compared with those obtained from muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.     

Pyruvate kinase isozymic shifts of differentiating chick myogenic cells in vivo and in culture.

Developing chick skeletal muscle undergoes an isozymic shift from type K pyruvate kinase to type M during development. A major increase in pyruvate kinase activity follows the isozymic shift, resulting in at least 40-fold higher specific activities by adulthood. Similar isozymic changes occur in primary cultures of myogenic cells from 12-day-old chick embryos. Cultures initially contain only type K pyruvate kinase. Type M appears by the fourth day of culture and accounts for 80–90% of the activity by the eleventh day. Type M did not accumulate when cell fusion was prevented by removing Ca²⁺ from the growth medium or when protein synthesis was inhibited by cycloheximide.     

Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.     

Conformational changes required for pyruvate kinase activity as modulated by monovalent cations.

The interaction of a series of alkylamines with muscle pyruvate kinase was investigated by kinetic and physical studies in order to understand the mechanisms by which certain monovalent cations can activate the enzyme and to define several of the important conformational changes necessary for catalytic activity. Monomethylammonium ion interacts with pyruvate kinase to activate the enzyme. Dimethyland trimethylammonium ions do not activate, but are competitive inhibitors against activating cations. Tetramethylammonium ion neither activates nor inhibits pyruvate kinase activity. When the enzyme is in the presence of monomethylammonium ion or dimethylammonium ion, a conformational change is observed by ultraviolet difference spectroscopy. This conformational change is similar to that observed with other activating cations and appears to be a necessary but no sufficient conformational change in the formation of an active complex. The interaction of the substrate phosphoenolpyruvate with the pyruvate kinase-Mn2+ complex in the presence of these cations was studied by water proton relaxation rate measurements. The affinity of the enzyme-Mn2+ complex for phosphoenolpyruvate is decreased by a factor of 5 in the presence of any of the alkylamines compared to the affinity measured in the presence of K+ or NH4+. No change in the Km of phosphoenolpyruvate is observed however when it is measured in the presence of monomethylammonium ion, suggesting that the decrease in affinity for the substrate is not the reason for lack of enzymic activity. The conformation of the ternary enzyme-Mn2+-phosphoenolpyruvate complex about the bound Mn2+, as reflected by the enhancement values (epsilont) measured, differs depending upon the nature of the monovalent cation. The epsilon t values measured in the presence of the alkylamines are larger (epsilont - 5.7 +/- 0.2) than those measured in the presence of K+ or NH4+ (epsilont = 1.9 +/- 0.1).     

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.