Synergistic effects of proton and phenylalanine on the regulation of muscle pyruvate kinase

Steady-state kinetic studies of muscle pyruvate kinase were conducted as a function of pH and phenylalanine concentrations. Results show that at a pH below 7.0, there is no observable effect of phenylalanine on the kinetic properties of muscle pyruvate kinase. When the results at a pH below 6.5 are used as the state for comparison, the kinetic results show that phenylalanine and proton exert a synergistic effect on the allosteric properties of the enzyme. A significantly greater change in Hill coefficients at high pH can be detected in the presence of phenylalanine than in its absence. To pinpoint the specific mechanism that leads to the synergistic effect, the kinetic data were resolved into the five equilibrium and two rate constants that characterize the basic two-state model. It can be shown that KTI, the binding constant of phenylalanine to the inactive T state, is strongly proton-linked. The affinity of phenylalanine for the T state increases with increasing pH. When the pH dependence of KTI was analyzed by the linked-function theory [Wyman, J. (1964) Adv. Protein Chem. 19, 224-285], it was shown that deprotonation favors phenylalanine binding to the T state. KRI (the binding constant of phenylalanine to the active R state), KTS (the binding constant of substrate to the T state), and L (the isomerization constant of the two states) not only are all weakly proton-linked but also it was shown that protonation favors the ligand-pyruvate kinase complex. KRS, the binding constant of substrate for the R state, shows no observable linkage to proton concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

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].     

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].     

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.     

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.     

Kinetic and mechanism of interaction of L-cysteine with alkaline phosphatase from calf intestines

The effect of L-cysteine on activity of hydrophobic forms of calf intestine alkaline phosphatase was investigated. Apparent inhibition constants for mixed type inhibition have been determined. The kinetic results allow supposing that the mechanism of equilibrium establishment between the inhibitor and enzyme involves the initial rapid formation of intermediate complex and a subsequent slower step leading to its stabilization in the substrate binding site. The microscopic rate constants for slow step of interaction of L-cysteine with alkaline phosphatase have been calculated. Effect of pH on apparent inhibition constants and kinetic parameters for enzymatic reaction in the presence of L-cysteine was analysed.     

Phosphorylation of pyruvate kinase type K is restricted to the dimeric form.

In the absence of glycolytic intermediate, fructose-1,6-bisphosphate, pyruvate kinase type K exists in the dimeric form and is readily phosphorylated, whereas in the same sample and the same conditions pyruvate kinase type M is present as a tetramer and is not phosphorylated. Addition of fructose-1,6-bisphosphate results in the association of dimeric K2 molecules to a tetrameric K4 enzyme as determined by gel filtration and cellulose acetate electrophoresis, with concomitant loss of the capacity of the K isozyme to become phosphorylated. Phosphorylated K2 dimers can also tetramerize, but with a low recovery of the radiolabel, suggesting a fructose-1,6-bisphosphate induced dephosphorylation or selective degradation. The dimeric K isozyme is enzymatically active; inactive K-type monomers can be detected by immunoblot analysis in the absence of fructose-1,6-bisphosphate, but no phosphorylated pyruvate kinase is present in this fraction. The formation of K4 tetramers can not be accomplished by the substrate phosphoenolpyruvate. Fructose-1,6-bisphosphate is an allosteric activator of pyruvate kinase type K and induces hyperbolic saturation curves for phosphoenolpyruvate. In contrast, in the absence of effectors, pyruvate kinase type M exhibits Michaelis-Menten kinetics, but sigmoidal curves can be induced by the amino acid phenylalanine. However, even in the presence of phenylalanine, the M-type maintained its tetrameric configuration and did not serve as a substrate in the phosphorylation reaction. These findings argue for the importance of subunit interaction in the regulation of phosphorylation of pyruvate kinase.     

A kinetic study of rabbit muscle pyruvate kinase

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg(2+), pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg(2+), form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.     

The kinetic effects of oxalate on liver and erythrocyte pyruvate kinases

Oxalate dianion exerts a dual effect on allosteric liver and erythrocyte pyruvate kinases. In the absence of fructose 1,6 bisphosphate and at phosphoenolpyruvate concentrations lower than K_0.5s, oxalate apparently behaves as an allosteric activator. In the presence of fructose 1,6 bisphosphate and at higher phosphoenolpyruvate concentrations, oxalate is a powerful competitive inhibitor with respect to phosphoenolpyruvate. Such properties are consistent with the allosteric model of Monod-Wyman-Changeux for a substrate analogue. Inhibition constants of oxalate towards pyruvate kinase are in the same order of magnitude as blood oxalate concentration.     

An immunological study of the interaction of ligands with pyruvate kinase of Neurospora crassa

Antibodies against pyruvate kinase of Neurospora crassa, induced in rabbits, were used to monitor the interaction of ligands with this enzyme. The technique of microcomplement fixation was employed to probe for conformational alterations elicited by binding of substrates (phosphoenolpyruvate (PEP) and adenosine diphosphate), the allosteric activator (fructose 1,6-diphosphate), and the inhibitor (valine). On binding of PEP and valine to pyruvate kinase a pronounced reduction in the extent of complement fixation was observed. The second substrate, ADP, had no effect while FDP elicited a moderate suppression of complement fixation. These results suggest that as a consequence of conformational changes induced by PEP and valine, some antigenic determinants on the surface of pyruvate kinase are rendered inaccessible to the antibodies.     

Allosteric kinetics of pyruvate kinase of Saccharomyces carlsbergensis

The allosteric model of Monod et al. (1965) has been used to analyse the steadystate kinetics of pyruvate kinase from Saccharomyces carlsbergensis. The dissociation constants for the substrate phosphoenolpyruvate, the inhibitor ATP as well as the activator fructose-1, 6-diphosphate from the R and T state were calculated using a series of computer programs. On the basis of a crucial relation (derived in the Appendix), which correlates the Hill coefficient and the half-saturating concentration of substrate saturation curves with the parameters of the model of Monod et al., it is possible to differentiate between exclusive and non-exclusive ligand binding. On the other hand, this relation makes it possible to fit the experimental data to an extended model assuming only partially concerted transitions in each enzyme molecule.The physical data of yeast pyruvate kinase point to a tetrameric structure, whereas the steady-state kinetics favour a trimeric one. This discrepancy in the number of protomers can be overcome by the use of an extended model, which permits the occurrence of hybrid states R_tT_n?t. The introduction of one symmetrical hybrid state R₂T₂ into the model explains the kinetic data of yeast pyruvate kinase on the basis of four, probably identical, protomers. The equilibrium constants between the states are given.In the Appendix the derivation of the equation describing the occurrence of hybrid states is reported.     

Kinetic properties of pyruvate kinase of rabbit brain

Summary The kinetic properties of rabbit brain pyruvate kinase have been studied to determine its role in the regulation of glycolysis. One of the substrates of the enzyme, phosphoenolpyruvate, exhibits homotropic cooperativity (Hill coeff. of 1.45); thus, it is a moderate activator of the enzyme. The other substrate, ADP, shows normal Michaelis-Menton kinetics. Fructose-6-phosphate and glucose-6-phosphate activate the enzyme only slightly at the 1mm level and inhibit slightly at higher levels, and hence have no metabolic influence on the enzyme activity. Fructose-1, 6-diphosphate also has a slight activation up to 0.5 mm but no inhibition at higher level; therefore, it has no influence either. ATP, 2-phosphoglycerate, and phenylalanine are inhibitors of the enzyme. ATP, being the energy reservoir derived from glycolysis as well as a product of the reaction catalyzed by the enzyme, is a significant feedback inhibitor of the enzyme. These kinetic properties suggest a key role for pyruvate kinase in the regulation of glycolysis. Phenylalanine inhibition of the enzyme has been reported to be a possible mechanism of damage to the developing brain in phenylketonuria. The inhibition by phenylalanine at 10 mm in the assay mixture is reversed by alanine, cysteine, or serine at 0.2 mm level. Furthermore, the effect of these amino acids in reversing the phenylalanine inhibition are mutually enhancing. Consequently phenylalanine cannot have a significant inhibition on the activity of pyruvate kinase in brain.A preliminary report has been presented at the American Society of Biological Chemists Meeting at Atlanta, Georgia, June 1978.     

Stereoselectivity of interaction of phosphoenolpyruvate analogues with various phosphoenolpyruvate-utilizing enzymes

The halogenated phosphoenolpyruvate analogues (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate, and (Z)-phosphoenol-3-bromopyruvate were synthesized and purified. The analogues were characterized by 1H and by 19F NMR where applicable. Absolute stereoselectivity of the fluorophosphoenolpyruvate isomers as substrates with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate phosphate dikinase was observed. The Z isomer exhibited substrate activity with these enzymes while no substrate activity was measured with the E isomer. Both isomers exhibited substrate activity with the enzyme pyruvate kinase, however, with a substantial decrease in the Vmax/Km ratio compared to phosphoenolpyruvate as the substrate. A metal ion dependent stereoselectivity of inhibition was measured for these analogues with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate kinase. The cation activator appears to affect the specificity and thus the catalytic site of these enzymes. Proton longitudinal relaxation rate titrations demonstrate that the dissociation constants, K3, of the fluorophosphoenolpyruvate isomers from the enzyme-Mn complex agree, in most cases, with the measured KI values and analogue binding resembles phosphoenolpyruvate binding. With the enzyme phosphoenolpyruvate carboxykinase, the KI not equal to K3 for (E)-fluorophosphoenolpyruvate which suggests that the binding of the E isomer is affected by the presence of the other substrates. The halogenated derivatives apparently undergo an enzyme-Mn catalyzed Michael-type addition reaction with the bromo-substituted analogue decomposing much faster than the fluoro analogues.     

Calorimetric demonstration of the potential of molecular crowding to emulate the effect of an allosteric activator on pyruvate kinase kinetics

A method based on isothermal calorimetry is described for the direct kinetic assay of pyruvate kinase. In agreement with earlier findings based on the standard coupled assay system for this enzyme in the presence of a fixed ADP concentration, the essentially rectangular hyperbolic dependence of initial velocity upon phosphoenolpyruvate concentration is rendered sigmoidal by the allosteric inhibitor phenylalanine. This effect of phenylalanine can be countered by including a high concentration of a space-filling osmolyte such as proline in the reaction mixtures. This investigation thus affords a dramatic example that illustrates the need to consider potential consequences of thermodynamic nonideality on the kinetics of enzyme reactions in crowded molecular environments such as the cell cytoplasm.     

The regulatory properties of yeast pyruvate kinase. Effects of NH4+ and K+ concentrations.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C and pH 6.2 as a function of the concentrations of ADP, phosphoenolpyruvate, Mg2+ and either NH4+ or K+. The data were analysed by the exponential model for four substrates, obtained by extension of the model described by Ainsworth, Kinderlerer & Gregory [(1983) Biochem. J. 209, 401-411]. On that basis, it was concluded that NH4+ binding is almost non-interactive but leads to the appearance of positive interaction in the velocity response to increase in its concentration because of positive interactions with phosphoenolpyruvate and Mg2+. The data obtained with K+ lead to the same conclusions and differ only in suggesting that NH4+ is bound more strongly to the enzyme than is K+. Both data sets are used as the basis for a discussion of the substrate interactions of pyruvate kinase and it appears therefrom that the heterotropic interactions accord with what is known of the events that take place at the active site during catalysis. The paper also reports a determination of the dissociation constants for the NH4+ complexes with ADP and phosphoenolpyruvate and an examination of the simultaneous activation of pyruvate kinase by K+ and NH4+ ions.     

Domain interaction in rabbit muscle pyruvate kinase. I. Effects of ligands on protein denaturation induced by guanidine hydrochloride

The structural stability of rabbit muscle pyruvate kinase was examined. The unfolding of pyruvate kinase was induced by guanidine hydrochloride, and the process was monitored by spectroscopic techniques (fluorescence and UV absorption) and hydrodynamic measurements (sedimentation velocity, sedimentation equilibrium, densimetry, and viscometry). The spectroscopic techniques revealed that the unfolding of pyruvate kinase induced by guanidine hydrochloride is not a simple cooperative process. This suggests that different regions of pyruvate kinase are unfolding with different efficiencies in response to the denaturant. These regions are most likely related to the domain structures observed by x-ray crystallography. In the presence of L-phenylalanine, the allosteric inhibitor, the denaturation process became more cooperative, and the enzyme dissociated and unfolded at a higher denaturant concentration. The binding of phenylalanine also induced a structural change in the enzyme, rendering it more susceptible to tryptic digestion. One of the peptides, the production rate of which was increased, was isolated and sequenced. Its N terminus is located at the interface between two domains, one of which contains the active site. This evidence indicates structural changes, probably involving domain-domain interaction, for pyruvate kinase in response to phenylalanine binding.     

Pyruvate kinase isozymes from the green alga, Selenastrum minutum. II. Kinetic and regulatory properties

The kinetic and regulatory properties of two pyruvate kinase isozymes, PKp and PKc (apparent chloroplastic and cytosolic isozymes, respectively) from the green alga Selenastrum minutum were studied. The two isozymes differed greatly in several kinetic properties. Although both isozymes showed hyperbolic substrate saturation kinetics, the apparent Michaelis constants for PEP and ADP were about twofold and fourfold lower, respectively, for PKc as compared with PKp. ADP was the preferred nucleotide substrate for both isozymes. However, PKc utilized alternate nucleotides far more effectively than did PKp. PKc and PKp also differed strongly in the effect of activators and inhibitors on the enzymes. Although both isozymes were activated by dihydroxyacetone phosphate (DHAP) with a similar activation constant of about 30 microM, this activator (0.5 mM) caused an approximate 30% increase in the Vmax of PKc, but had no effect on the Vmax of PKp. PKp, but not PKc, was inhibited by ribose 5-phosphate, ribulose 1,5-bisphosphate, 2-phosphoglycerate, phosphoglycolate, and malate. Both isozymes were inhibited by MgATP, Mg2citrate, Mg2oxalate, and Pi. PKc was far more sensitive to inhibition by Pi, as compared with PKp. Pi was a competitive inhibitor of PKc with respect to phosphoenolpyruvate (PEP) (Ki = 1.3 mM). Glutamate was a potent inhibitor of PKc, but had no effect on PKp. In contrast with Pi, glutamate was a mixed-type inhibitor of PKc with respect to PEP (Ki = 0.7 mM). DHAP facilitated the binding of PEP by both isozymes and reversed or relieved the inhibition of PKc by Pi and/or glutamate. The regulatory properties of PKp indicate that it is likely less active in the light and more active in the dark. The in vivo activity of PKc is probably regulated by the relative cytosolic levels of DHAP, Pi, and glutamate; this provides a rationale for the activation of algal cytosolic pyruvate kinase which occurs during periods of enhanced ammonia assimilation.     

Regulation of pyruvate kinases from Fusarium oxysporum.

Two types of pyruvate kinases were found in Fusarium oxysporum. One type (inducible) was present mainly during the early stages of growth on glucose or sucrose and displayed Michaelis-Menten kinetics with respect to phosphoenolpyruvate and adenosine diphosphate. The major type (constitutive) was present under all conditions of growth and displayed in the absence of potassium ions, a sigmoidal substrate saturation curve when phosphoenolpyruvate was used as the variable substrate. In the presence of potassium ions the saturation curve for phosphoenolpyruvate exhibits a plateau at half-maximal velocity. The effects of various metabolites on the activity of the inducible and constitutive kinases were also studied. Fructose-1,6-diphosphate, cyclic AMP, acetyl CoA, tryptophan, and phenylalanine had no effect on the activity of the enzymes. Citrate was a potent inhibitor of the constitutive pyruvate kinase activity and increased the sigmoidicity of the saturation curve for phosphoenolpyruvic acid. In the presence of K+, the bimodal plot observed in the absence of citrate gradually changed to a hyperbolic shape as the concentration of citric acid was increased. In the presence of K+ and ADP as the variable substrate citric acid converted the hyperbolic plot to a sigmoidal one. Citrate had no effect on the inducible enzyme.     

Hemolytic anemia due to pyruvate kinase deficiency: characterization of the enzymatic activity from eight patients.

We have studied the red cell pyruvate kinase (PK) variants from eight patients representing five families with pyruvate kinase deficiency-associated hemolytic anemia. The kinetic properties, electrophoretic mobilities, and immunological reactivity with anti-normal red cell pyruvate kinase were determined. The patients differ in the severity of their clinical condition and in the molecular properties of their red cell pyruvate kinase variants. The most seriously affected patient (PK Beaverton) has no electrophoretically demonstrable red cell isozymes. The activity present is due to the M2 isozyme, however red cell isozyme can be detected immunologically. PK Molalla and PK Lake Oswego are thermolabile variants with normal kinetic parameters. PK Molalla, in addition, has altered electrophoretic mobility. PK Multnomah and PK Milwaukie have decreased affinity for the substrate phosphoenolpyruvate, and PK Multnomah also has altered electrophoretic mobility. PK Coos Bay shows electrophoretic variation and a slightly decreased affinity for phosphoenolpyruvate consistent with an increased modulating effect of fructose-1,6-diphosphate.     

Synthesis and study of (Z)-3-chlorophosphoenolpyruvate

(Z)-3-Chlorophosphoenolpyruvate has been synthesized by the reaction of 3,3-dichloropyruvic acid with trimethylphosphite, followed by deesterification. This compound is a competitive inhibitor of pyruvate kinase and phosphoenolpyruvate carboxylase. Pyruvate kinase is not inactivated upon prolonged incubation with the compound, but phosphoenolpyruvate carboxylase is slowly inactivated (t1/2 = 5 h). The compound is a substrate for both enzymes, being acted upon by pyruvate kinase approximately 0.1% as rapidly as phosphoenolpyruvate itself. In the case of phosphoenolpyruvate carboxylase, the compound is converted into a 3:1 mixture of chloropyruvate and chlorooxalacetate, at an overall rate that is about 25% the carboxylation rate for phosphoenolpyruvate.