Energetics of allosteric regulation in muscle pyruvate kinase.

The regulatory mechanism of rabbit muscle pyruvate kinase has been studied as a function of temperature in conjunction with phenylalanine, the allosteric inhibitor. The inhibitory effect of phenylalanine is modulated by temperature. At low temperatures, the presence of phenylalanine is almost inconsequential, but as the temperature increases so does the phenylalanine-dependent inhibition of the kinetic activity. In addition, the presence of phenylalanine induces cooperativity in the relation between velocity and substrate concentration. This effect is especially pronounced at elevated temperature. The kinetic data were analyzed using an equation that describes the steady-state kinetic velocity data as a function of five equilibrium constants and two rate constants. Van't Hoff analysis of the temperature dependence of the equilibrium constants determined by nonlinear curve fitting revealed that the interaction of pyruvate kinase with its substrate, phosphoenolpyruvate, is an enthalpy-driven process. This is consistent with an interaction that involves electrostatic forces, and indeed, phosphoenolpyruvate is a negatively charged substrate. In contrast, the interaction of pyruvate kinase with phenylalanine is strongly entropy driven. These results imply that the binding of phenylalanine involves hydrophobic interaction and are consistent with the basic concepts of strengthening of the hydrophobic effect with an increase in temperature. The effect of phenylalanine at high temperatures is the net consequence of weakening of substrate-enzyme interaction and significant strengthening of inhibitor binding to the inactive state of pyruvate kinase. The effects of salts were also studies. The results show that salts also exert a differential effect on the binding of substrate and inhibitor to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of pH-dependence of kinetic parameters of pyruvate kinase from bovine adrenal cortex

The effect of pH on the main kinetic parameters of pyruvate kinase function was studied. The maximal rate of the reaction as well as the values of Km for ADP and Ki for phenylalanine depend on pH and show a well-defined extremum at pH 6.8-7.0. Spectrofluorimetric titration of pyruvate kinase results in pH dependencies of changes in the fluorescence spectra parameters (e.g., quantum yield, half-width and position of the maximum). This enabled to determine the pH regions corresponding to changes in the state of tryptophan residues. Data from the enzyme inhibition by phenylalanine suggest that acidification of the medium leads to the decrease of the catalytic activity due to the protonation of the ionogenic group of the enzyme. Within the pH range of 7.0-8.0, the decrease of the pyruvate kinase activity is due to structural shifts in the enzyme molecule, as a result of which the steric complementariness of the enzyme active center with respect to the substrate (Mg.ADP) is impaired.     

The purification and kinetic characterization of eel white muscle pyruvate kinase

A stable, homogeneous preparation of pyruvate kinase from white muscle of the American eel, Anguilla rostrata with a specific activity of 350 units/mg has been obtained. The enzyme has a pH optimum in the range 6.3-6.5 and requires Mg2+ and K+ for maximum activity. Eel muscle pyruvate kinase exhibits slight co-operativity in the binding of the substrate phosphoenol-pyruvate. It is activated by fructose-1,6-bisphosphate in a pH dependent manner and is inhibited by both alanine and phenylalanine. These properties are very similar to the properties of the mammalian M2 isozyme.     

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.     

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.     

A tentative mechanism of the ternary complex formation between phosphorylase kinase, glycogen phosphorylase b and glycogen

The kinetics of rabbit skeletal muscle phosphorylase kinase interaction with glycogen has been studied. At pH 6.8 the binding of phosphorylase kinase to glycogen proceeds only in the presence of Mg2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca2+ at both pH values. The initial rate of the complex formation is proportional to the enzyme and glycogen concentrations, suggesting the formation of the complex with stoichiometry 1:1 at the initial step of phosphorylase kinase binding by glycogen. According to the kinetic and sedimentation data, the substrate of the phosphorylase kinase reaction, glycogen phosphorylase b, favors the binding of phosphorylase kinase with glycogen. We suggest a model for the ordered binding of phosphorylase b and phosphorylase kinase to the glycogen particle that explains the increase in the tightness of phosphorylase kinase binding with glycogen in the presence of phosphorylase b.     

Thermodynamic studies of the activation of rabbit muscle lactate dehydrogenase by phosphate.

In an attempt to trace the source of phosphate activation of the enzyme-catalysed pyruvate-lactate interconversion by rabbit muscle lactate dehydrogenase, equilibrium constants were measured to examine the effects of phosphate on interactions pertinent to the enzymic process. Frontal gel-chromatographic studies of the binding of NADH to the enzyme established that the intrinsic association constant is doubled in the presence of 50 mM-phosphate in the buffer (pH 7.4, I0.15). From kinetic studies of the competition between NAD+ and NADH for the coenzyme-binding sites of the enzyme it is concluded that the binding of oxidized nicotinamide nucleotide is also doubled in the presence of 50 mM-phosphate. Competitive-inhibition studies and fluorescence-quenching measurements indicated the lack of a phosphate effect on ternary-complex formation between enzyme-NADH complex and oxamate, a substrate analogue of pyruvate. The equilibrium constant for the interaction between enzyme-NAD+ complex and oxalate, an analogue of lactate, was also shown, by difference spectroscopy, to be insensitive to phosphate concentration. Provided that the effects observed with the substrate analogues mimic those operative in the kinetic situation, the equilibrium constant governing the isomerization of ternary complex is also independent of phosphate concentration. It is concluded that enhanced coenzyme binding is the source of phosphate activation of the rabbit muscle lactate dehydrogenase system.     

Thermodynamic linkages in rabbit muscle pyruvate kinase: kinetic, equilibrium, and structural studies

The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].     

Creatine kinase equilibrium and lactate content compared with muscle pH in tissue samples obtained after isometric exercise

Muscle biopsies taken from the musculus quadriceps femoris of man were analysed for pH, ATP, ADP, AMP, creatine phosphate, creatine, lactate and pyruvate. Biopsies were taken at rest, after circulatory occlusion and after isometric contraction. Muscle pH decreased from 7.09 at rest to 6.56 after isometric exercise to fatigue. Decrease in muscle pH was linearly related to accumulation of lactate plus pyruvate. An increase of 22mumol of lactate plus pyruvate per g of muscle resulted in a fall of 0.5pH unit. The apparent equilibrium constant of the creatine kinase reaction (apparent K(CK)) increased after isometric contraction and a linear relationship between log(apparent K(CK)) and muscle pH was obtained. The low content of creatine phosphate in muscle after contraction as analysed from needle-biopsy samples is believed to be a consequence of an altered equilibrium state of the creatine kinase reaction. This in turn is attributed mainly to a change in intracellular pH.     

Thermodynamic nonideality as a probe of allosteric mechanisms: preexistence of the isomerization equilibrium for rabbit muscle pyruvate kinase

Sedimentation velocity studies in the presence and absence of an inert space-filling solute, sucrose, have been used to establish preexistence of the isomerization equilibrium responsible for the allosteric behavior of rabbit muscle pyruvate kinase. Whereas the inclusion of phenylalanine (5 mM) with enzyme gives rise to a decrease of 0.3 S in the sedimentation coefficient of pyruvate kinase, the corresponding effect of phosphoenolpyruvate is to increase the sedimentation coefficient by 0.03 S. Consideration of these findings to signify the existence of an isomeric equilibrium between compact and expanded forms of the enzyme is substantiated by the finding that inclusion of sucrose (0.1 M) also brings about the change in sedimentation coefficient effected by phosphoenolpyruvate. By demonstrating that rabbit muscle pyruvate kinase undergoes isomerization in the absence of substrate, this study removes any necessity to consider the existence of an isomerization equilibrium that is substrate-induced; and thereby provides experimental support for adoption of the Monod model of allostery to interpret enzyme kinetic data for pyruvate kinase [R. W. Oberfelder, B. G. Barisas, and J. C. Lee (1984) Biochemistry 23, 3822-3826].     

The proton transfer step catalyzed by yeast pyruvate kinase

The nature of the proton donor to the C-3 of the enolate of pyruvate, the intermediate in the reaction catalyzed by yeast pyruvate kinase, was investigated by site-directed mutagenesis and physical and kinetic analyses. Thr-298 is correctly located to function as the proton donor. T298S and T298A were constructed and purified. Both mutants are catalytically active with a decrease in k(cat) and k(cat)/K(m)(,PEP). Mn(2+)-activated T298S and T298A do not exhibit homotropic kinetic cooperativity with phosphoenolpyruvate (PEP) in the absence of fructose 1,6-bisphosphate, although PEP binding to enzyme-Mn(2+) is cooperative. The pH dependence of k(cat) for T298A indicates the loss of pK(a)(,2) = 6.4-6.9. Thr-298 affects the ionization (pK(a) approximately 6.5) responsible for modulation of k(cat). Fluorescence studies show altered dissociation constants of ligands to each enzyme complex upon Thr-298 mutations. The rates of the phosphoryl transfer and proton transfer steps in the pyruvate kinase-catalyzed reaction are altered; pyruvate enolization is affected to a greater extent. Proton inventory studies demonstrate solvent isotope effects on k(cat) and k(cat)/K(m)(,PEP). Fractionation factors are metal-dependent and significantly <1. The data suggest that a water molecule in a water channel is the direct proton donor to enolpyruvate and that Thr-298 affects a late step in catalysis.     

The reversibility of skeletal muscle pyruvate kinase and an assessment of its capacity to support glyconeogenesis.

The kinetics of pyruvate phosphorylation by rabbit skeletal muscle pyruvate kinase (EC 2.7.1.40) has been studied with a coupled assay using P-enolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). The reaction sequence is (See journal for formula). Although the equilibrium of the pyruvate kinase reaction by itself strongly favors pyruvate production, the over-all equilibrium of this coupled system favors the depletion of pyruvate, thus greatly reducing the problem of back reaction during the assay. In addition, the oxidation of NADH by malate dehydrogenase makes it possible to monitor the system with a spectrophotometer. The Michaelis constant of pyruvate kinase was found to be 0.9 mM for ATP and 7 mM for pyruvate, values that agree reasonably well with earlier studies using direct assays. However, the maximum velocity is about 6 mumol of pyruvate phosphorylated/min/mg of enzyme, which is very much faster than that indicated by earlier studies. These results suggest that the metabolic significance of the reverse reaction of muscle pyruvate kinase may have been underestimated. In particular, the data given here suggest that its rate in vivo is probably comparable to the observed rate of glycogen synthesis from lactate, making possible glyconeogenesis in muscle by pyruvate kinase reversal without the need for an enzymatic bypass of the kind employed by liver and kidney.     

Regulation of thyroid hormone binding to its cytosolic binding protein by L-alpha-alanine

The human cytosolic thyroid hormone binding protein (p58) was recently shown to be a monomer of pyruvate kinase, subtype PKM2, and have intrinsic pyruvate kinase activity. The present study evaluated the effect of L-alpha-alanine on the binding of 3,3',5-triiodo-L-thyronine (T3) and enzymatic activity of p58. Analysis of the competitive binding data indicated that alanine, at the physiological concentration, is a non-competitive inhibitor of T3 binding to p58. Furthermore, alanine was found to be a "mixed" inhibitor of the substrate phosphoenol pyruvate. However, binding of alanine to p58 did not block the association of p58 to form the tetrameric pyruvate kinase.     

Octopine dehydrogenase from Pecten maximus: steady-state mechanism

The steady-state kinetic mechanism of the reaction catalyzed by octopine dehydrogenase [N2-(1-carboxyethyl)-L-arginine:NAD+ oxidoreductase] was investigated at pH 6.9 and 9.2 by studies of substrate inhibition, analogue inhibition, and product inhibition. In the direction of octopine synthesis, the inhibition patterns in the presence of delta- guanidinovalerate and propionate show that NADH binds to the enzyme first followed by L-arginine and pyruvate which bind randomly. In the direction of octopine oxidation, the substrate patterns show that NAD binds to the enzyme before octopine in a rapid equilibrium fashion, and the product inhibition patterns show that the products L-arginine and pyruvate are released in a random fashion. Double, synergistic, substrate inhibition by L-arginine and pyruvate was shown to be due to binding (hypothetically of the imine) to the free enzyme and the enzyme-NAD complex. Furthermore, an alternate minor pathway was demonstrated which includes an enzyme-NADH-octopine complex and an enzyme-octopine complex.     

The proton transfer reactions catalyzed by yeast pyruvate kinase.

1. The proton-transfer reactions of yeast pyruvate kinase (EC 2.7.1.40) were studied. Proton-transfer from C-3 of phosphoenolpyruvate to water occurs only in the presence of the phosphoryl-acceptor ADP. Proton transfer from C-3 of pyruvate to water occurs only in the presence of ATP. However, the proton transfer in the latter case occurs 10-100 times faster than phosphoryl transfer; this supports a mechanism in which proton transfer precedes phosphoryl transfer in the reverse reaction of pyruvate kinase. 2. The characteristics of proton-transfer reactions of yeast pyruvate kinase were compared with those previously reported for rabbit muscle pyruvate kinase (Robinson, JL. and Rose, I.A. (1972) J. Biol. Chem. 247, 1096-1105). The pH-profiles and the divalent cation dependencies were similar for Fru-1,6-P2-activated yeast pyruvate kinase and the muscle enzyme. Pyruvate enolization by yeast pyruvate kinase has an absolute requirement for ATP in contrast to enolization by the muscle enzyme which proceeds when ATP is replaced by Pi or other dianions. 3. Fructose-1,6-bisphosphate was shown to affect the catelytic steps of yeast pyruvate kinase in addition to the binding of substrates. Its role depends on the divalent cation used to activate the enzyme.     

pH studies on the chemical mechanism of rabbit muscle pyruvate kinase. 2. Physiological substrates and phosphoenol-alpha-ketobutyrate

pH profiles have been determined for the reactions catalyzed by pyruvate kinase between pyruvate and MgATP and between phosphoenolpyruvate and MgADP. V, V/KMgATP, and V/Kpyruvate all decrease below a pK of 8.3 and above one of 9.2. The group with pK = 8.3 is probably a lysine that removes the proton from pyruvate during enolization, while the pK of 9.2 is that of water coordinated to enzyme-bound Mg2+. The fact that this pK shows in all three pH profiles shows that pyruvate forms a predominantly second sphere complex and cannot replace hydroxide to form the inner sphere complex that results in enolization and subsequent phosphorylation. On the basis of the displacement of the pK of the acid-base catalytic group in its V/K profile, phosphoenolpyruvate is a sticky substrate, reacting to give pyruvate approximately 5 times faster than it dissociates. The V/K profile for the slow substrate phosphoenol-alpha-ketobutyrate shows the pK of 8.3 for the acid-base catalytic group in its correct position, but this group must be protonated so that it can donate a proton to the intermediate enolate following phosphoryl transfer. The secondary phosphate pK of the substrate is seen in this V/K profile as well as in the pKi profile for phosphoglycolate (but not in those for glycolate O-sulfate or oxalate), showing a preference for the trianion for binding. The chemical mechanism with the natural substrates thus appears to involve phosphoryl transfer between MgADP and a Mg2+-bound enolate with metal coordination of the enolate serving to make it a good leaving group.     

The cooperative binding of fructose-1,6-bisphosphate to yeast pyruvate kinase.

The cooperative binding of the allosteric activator fructose-1,6-bisphosphate [Fru(1,6)P2] to yeast pyruvate kinase was investigated by equilibrium dialysis and fluorescence quench titration. The results show that yeast pyruvate kinase binds four molecules of Fru(1,6)P2 per tetramer and the observed fluorescence quench follows the binding of the ligand and not the cooperative T to R state transition. Additionally it is shown that the binding of Fru(1,6)P2 to yeast pyruvate kinase is compatible with the model of cooperativity that has been proposed and incorporates an intermediate state, R', with properties between those of the T and R states.     

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     

An investigation of heavy meromyosin-ADP binding equilibria by proton release measurements.

The interaction of magnesium-ADP with skeletal muscle heavy meromyosin has been studied by measuring the accompanying release of protons. Total pH changes of the order of 0.03 were involved, and measurements were performed with a discrimination of some ten-thousandths of a pH unit. At pH 8.0 and 25 degrees C about 0.5 mol of protons per mol of heavy meromyosin is released at saturation. A stoichiometry of binding close to 2 mol of ADP per mol of protein was found, with a binding constant, obtained from the proton release titration curve (pH 8.0, 25 degrees C), of 2 X 10(5) M-1. At 5 degrees C the release of protons per mole is slightly greater, and the binding constant is somewhat increased, reflecting a negative enthalpy of binding. Similar proton release behavior is observed in the presence of manganous ions in place of magnesium. The liberation of protons is thus unrelated to the temperature-dependent isomerization of myosin in the presence of substrate. Alkylation of a reactive thiol group (SH1) does not change the proton liberation at pH 8.0. From the pH dependence of proton release, the association constant of heavy meromyosin with magnesium-ADP at other pH values can be inferred and shows an appreciable rise as the pH increases. The pH-proton release profile also allows the pK of the ionizing groups perturbed by the ligand to be deduced. At least two groups ionizing above pH 7 and one below are involved. Their pK's in the unperturbed state are assigned as 8.5, 9.3, and about 6.6, respectively; they are displaced in the complex to about 8.0, 9.1, and 6.3. A relation to the pH-activity profile of myosin ATPase is indicated. The pH-proton release profile is somewhat changed when the SH1 group is alkylated. Measurements with potassium-ADP, in the absence of magnesium, show that at pH 8.0 there is no proton release but rather a sizeable proton absorption (about 0.5 mol of protons per mol of heavy meromyosin). The association constant derived from the titration curves (pH 8.0, 25 degrees C) is 3 X 10(4) M-1.     

Probing of the substrate binding domain of lactose permease by a proton pulse

The lactose permease of Escherichia coli coupled proton transfer across the bacterial inner membrane with the uptake of beta-galactosides. In the present study we have used the cysteine-less C148 mutant that was selectively labeled by fluorescein maleimide on the C148 residue, which is an active component of the substrate transporting cavity. Measurements of the protonation dynamics of the bound pH indicator in the time resolved domain allowed us to probe the binding site by a free diffusing proton. The measured signal was reconstructed by numeric integration of differential rate equations that comply with the detailed balance principle and account for all proton transfer reactions taking place in the reaction mixture. This analysis yields the rate constants and pK values of all residues participating in the fast proton transfer reaction between the bulk and the protein's surface, revealing the exposed residues that react with free protons in a diffusion controlled reaction and how they transfer protons among themselves. The magnitudes of these rate constants were finally evaluated by comparison with the rate predicted by the Debye-Smoluchowski equation. The analysis of the kinetic and pK values indicated that the protein-fluorescein adduct assumes two conformation states. One is dominant above pH 7.4, while the other exists only below 7.1. In the high pH range, the enzyme assumes a constrained configuration and the rate constant of the reaction of a free diffusing proton with the bound dye is 10 times slower than a diffusion controlled reaction. In this state, the carboxylate moiety of residue E126 is in close proximity to the dye and exchanges a proton with it at a very fast rate. Below pH 7.1, the substrate binding domain is in a relaxed configuration and freely accessed by bulk protons, and the rate of proton exchange between the dye and E126 is 100,000 times slower. The relevance of these observations to the catalytic cycle is discussed.