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.     

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)     

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.     

Kinetic properties of cerebral pyruvate kinase

Partly purified guinea-pig brain pyruvate kinase is not activated by fructose 1,6-diphosphate and gives hyperbolic substrate-saturation curves with phosphoenolpyruvate. It is therefore different from the L-type pyruvate kinase of mammalian liver. Inhibition by MgATP(2-) was competitive for MgADP(-) but not for phosphoenolpyruvate, and the enzyme is therefore different from the M-type pyruvate kinase, which is said to be competitively inhibited by MgATP(2-) with respect to both substrates. The K(i)(MgATP(2-)) value of approx. 8mm for the brain enzyme is higher than the values (about 2mm) reported for the muscle enzyme. Stimulation of enzymic activity was observed at low (1-2mm) concentrations of MgATP(2-). Substrate kinetic constants were K(m) (MgADP(-))=0.47mm, K(m) (phosphoenolpyruvate)=0.08mm. Free Mg(2+) at very high concentrations (over 10mm) was inhibitory (K(i)=20-32mm). Neither ADP(3-) nor 5'-AMP(2-) inhibited the activity. The brain enzyme was concluded to be different from both the M-type and the L-type of other mammalian organs such as muscle and liver.     

Kinetic properties of pig pyruvate kinases type A from kidney and type M from muscle.

Kinetic properties of homogeneous preparations of pig kidney and pig muscle pyruvate kinases (EC 2.7.1.40) were studied. Both isozymes showed a hyperbolic relationship to ADP with an apparent K_m of 0.3 mm. K⁺ and Mg²⁺ were necessary for the activity of both isozymes, and their dependences on these cations were similar. The muscle isozyme expressed Michaelis-Menten type of kinetics with respect to phosphoenolpyruvate, and the apparent K_m was the same (0.03 mm) from pH 5.5 to pH 8.0. In contrast, the dependence on phosphoenolpyruvate changed with pH for the kidney isozyme. It showed similar properties to the muscle isozyme at pH 5.5–7.0 (apparent K_m of 0.08 mm), while two apparent K_m values for this substrate were present at pH 7.5–8.0, one low (0.1 mm) and one high (0.3–0.6 mm). At pH 7.5, fructose 1,6-bisphosphate converted the kidney isozyme to a kinetical form where only the lower apparent K_m for phosphoenolpyruvate was detected. On the other hand, in the presence of alanine or phenylalanine the kidney pyruvate kinase showed only the higher K_m for this substrate. At low phosphoenolpyruvate levels both isozymes were inhibited by phenylalanine, and half-maximal inhibition was found at 0.3 and 2.2 mm for the kidney and muscle isozymes, respectively. At a 5 mm concentration of the substrate only the kidney isozyme was inhibited, the apparent K_i being the same. Alanine inhibited the kidney isozyme (apparent K_i at 0.3 mm, irrespective of substrate concentration). No effect was seen on the muscle isozyme. Fructose 1,6-bisphosphate was an activator of the kidney isozyme at phosphoenolpyruvate concentrations below 1.0 mm It also counteracted the inhibition by alanine or phenylalanine of this isozyme. ATP inhibited both isozymes, and this inhibition was not counteracted by fructose 1,6-bisphosphate. The kidney isozyme showed both a high and a low apparent K_m for phosphoenolpyruvate in the presence of ATP. The influence of the effectors on the activity of both isozymes varied markedly with pH, except for the action of ATP. At low substrate concentrations, however, the inhibitor action of ATP on the muscle enzyme was diminished around pH 7.5, in contrast to higher or lower pH values. Alanine or phenylalanine were more effective as inhibitors at higher pH values, and fructose 1,6-bisphosphate stimulated the kidney isozyme only at pH levels above pH 6.5. The influence of activators and inhibitors on the regulation of the kidney and muscle pyruvate kinases is discussed.     

The effect of sugar phosphates, phosphoenolpyruvate and adenylic acid on muscle, brain and heart creatine kinases]

The effect of adenylic acid, glucose-6-phosphate, fructose-1,6-diphosphate and phosphoenolpyruvate on creatine kinase isoenzymes (brain extract, muscle and heart extracts and purified muscle enzyme) was studied. These effectors, especially phosphoenolpyruvate, are shown to inhibit in different degree the reaction of ATP formation catalysed by creatine kinase from all tissues. The effectors do not inhibit the creatine phosphate synthesis in extracts, but depress purified creatine kinase. The interrelationship of the creatine kinase system and the key glycolytic enzymes (phosphofructokinase, hexokinase, pyruvate kinase) is discussed.     

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.     

Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13mum produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the K(m) values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn(2+) in addition to Mg(2+) for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.     

Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3', 5'-AMP-stimulated protein kinase.

The kinetics of rat liver L-type pyruvate kinase (EC 2.7.1.40), phosphorylated with cyclic AMP-stimulated protein kinase from the same source, and the unphosphorylated enzyme have been compared. The effects of pH and various concentrations of substrates, Mg2+, K+ and modifiers were studied. In the absence of fructose 1, 6-diphosphate at pH 7.3, the phosphorylated pyruvate kinase appeared to have a lower affinity for phosphoenolpyruvate (K0.5=0.8 mM) than the unphosphorylated enzyme (K0.5=0.3 mM). The enzyme activity vs. phosphoenolpyruvate concentration curve was more sigmoidal for the phosphorylated enzyme with a Hill coefficient of 2.6 compared to 1.6 for the unphosphorylated enzyme. Fructose 1, 6-diphosphate increased the apparent affinity of both enzyme forms for phosphoenolpyruvate. At saturating concentrations of this activator, the kinetics of both enzyme forms were transformed to approximately the same hyperbolic curve, with a Hill coefficient of 1.0 and K0.5 of about 0.04 mM for phosphoenolpyruvate. The apparent affinity of the enzyme for fructose 1, 6-diphosphate was high at 0.2 mM phosphoenolpyruvate with a K0.5=0.06 muM for the unphosphorylated pyruvate kinase and 0.13 muM for the phosphorylated enzyme. However, in the presence of 0.5 mM alanine plus 1.5 mM ATP, a higher fructose 1, 6-diphosphate concentration was needed for activation, with K0.5 of 0.4 muM for the unphosphorylated enzyme and of 1.4 muM for the phosphorylated enzyme. The results obtained strongly indicate that phosphorylation of pyruvate kinase may also inhibit the enzyme in vivo. Such an inhibition should be important during gluconeogenesis.     

Selective modification of rabbit muscle pyruvate kinase by 5-chloro-4-oxopentanoic acid.

Rabbit muscle pyruvate kinase was irreverisbly inactivated by 5-chloro-4-oxopentanoic acid with a pKa of 9.2. The inhibition was time-dependent and was related to the 5-chloro-4-oxopentanoic acid concentration. Analysis of the kinetics of inhibition showed that the binding of the inhibitor showed positive co-operativity (n = 1.5 +/- 0.2). Inhibition of pyruvate kinase by 5-chloro-4-oxopentanoic acid was prevented by ligands which bind to the active site. Their effectiveness was placed in the order Mg2+ greater than phosphoenolpyruvate greater than ATP greater than ADP greater than pyruvate. Inhibitor-modified pyruvate kinase was unable to catalyse the detritiation of [3-(3)H]pyruvate in the ATP-promoted reaction, but it did retain 5-10% of the activity with either phosphate or arsenate as promoters. 5-Chlor-4-oxo-[3,5-(3)H]pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoicheiometry of 1 mol of inhibitor bound per mol of pyruvate kinase protomer. The incorporation of the inhibitor and the loss of enzyme was proportional. These results are discussed in terms of 5-chloro-4-oxopentanoic acid alkylating a functional group in the phosphoryl overlap region of the active site, and a model is presented in which this compound alkylates an active-site thiol in a reaction that is controlled by a more basic group at the active site.     

Regulation of rat liver pyruvate kinase. The effect of preincubation, pH, copper ions, fructose 1,6-diphosphate and dietary changes on enzyme activity

1. Preincubation of partially purified rat liver L-type pyruvate kinase at 25 degrees for 10min. causes a marked increase in co-operativity with respect to both the substrate, phosphoenolpyruvate, and the allosteric activator, fructose 1,6-diphosphate. 2. The results are consistent with the existence of two forms of liver L-type pyruvate kinase, designated forms L(A) and L(B). It is postulated that form L(A) has a low K(m) for phosphoenolpyruvate (about 0.1mm) and is not allosterically activated, whereas form L(B) is allosterically activated by fructose 1,6-diphosphate, exhibiting in the absence of the activator sigmoidal kinetics with half-maximal activity at about 1mm-phosphoenolpyruvate. In the presence of fructose 1,6-diphosphate, form L(B) gives Michaelis-Menten kinetics with K(m) less than 0.1mm. It is further postulated that preincubation converts form L(A) into form L(B). 3. The influence of pH on the preincubation effect was studied. 4. The inhibition of pyruvate kinase by Cu(2+) was studied in detail. Though phosphoenolpyruvate and fructose 1,6-diphosphate readily protect the enzyme against Cu(2+) inhibition, little evidence of significant reversal of the inhibition by these compounds could be found. 5. The effects of starvation, fructose feeding and preincubation on the pyruvate kinase activity of crude homogenates of various tissues of the rat were also studied.     

The effect of fructose diphosphate and phosphoenolpyruvate on cyclic AMP-mediated inactivation of rat hepatic pyruvate kinase.

The addition of cyclic AMP and Mg-ATP to Sephadex-treated hepatocyte homogenates produced a time dependent inactivation of pyruvate kinase. The concentration of cyclic AMP giving half-maximal inhibition was 0.16 μM. The cyclic AMP-induced inactivation of pyruvate kinase was characterized by an increase in the K_0.5 for phosphoenolpyruvate from 0.56 to 1.15 mM and could be completely blocked by the addition of the protein kinase inhibitor. These experiments provide clear evidence that the cyclic AMP induced inactivation is a result of enzyme phosphorylation. Fructose-diphosphate and phosphoenolpyruvate, at physiological concentrations, suppressed inactivation induced by submaximal concentrations of cyclic AMP. It is suggested that hormonal induced changes in the levels of fructose diphosphate and phosphoenolpyruvate may influence the phosphorylation state of the enzyme in intact cells.     

Synthesis of oxalyl phosphate and processing of the acyl phosphate by phosphoenolpyruvate-dependent enzymes

The mixed anhydride of oxalic and phosphoric acids, oxalyl phosphate, has been prepared by reaction of oxalyl chloride and inorganic phosphate in aqueous solution. The product was purified by anion exchange chromatography and characterized by 31P and 13C NMR. This acyl phosphate has a half-life of 51 h at pH 5.0 and 4 degrees C. Oxalyl phosphate, an analogue of phosphoenolpyruvate, is a slow substrate for pyruvate kinase, undergoing an enzyme-dependent phosphotransfer reaction to produce ATP from ADP. Oxalyl phosphate substitutes for phosphoenolpyruvate in the reaction catalyzed by pyruvate, phosphate dikinase. The acyl phosphate reacts with the free enzyme to give the phosphorylated form of the enzyme. Removal of the potent product inhibitor, oxalate, from the reaction mixtures by gel filtration chromatography permitted further reaction of the phosphorylated enzyme with pyrophosphate and AMP to give ATP and Pi in a single turnover assay. Oxalyl phosphate also served as a phospho group donor in a partial reaction catalyzed by phosphoenolpyruvate carboxykinase wherein GDP is phosphorylated at the expense of oxalyl phosphate.     

Purification and kinetic properties of pyruvate kinase from Brochothrix thermosphacta.

Pyravate kinase (ATP: pyruvate 2-0 phosphotransferase E.C.2.7.1.40) was purified from Brochothrix thermosphacta. The enzyme is a homotetramer of monomer Mr 58,000. Fructose-1,6-bisphosphate stimulates activity and promotes hyperbolic kinetics although it is not essential for enzyme activity. The positive effect of fructose-1,6-bisphosphate on activity is repressed by inorganic phosphate which enhances cooperative kinetics. Unlike pyruvate kinases from other sources, the Brochothrix enzyme is uncompetitively inhibited by glucose-6-phosphate, although at high concentration. ATP is a strong inhibitor of pyruvate kinase and shifts the residual activity/pH profile towards more alkaline values.     

The internal pH and membrane potential of the insulin-secretory granule

1. Incubation of isolated hepatocytes with fructose at concentrations above 3 mM resulted in an apparent inhibition of pyruvate kinase assayed in crude extracts at sub-optimal phosphoenolpyruvate concentrations. 2. Fructose at concentrations below 3 mM caused an activation of the enzyme. 3. Increases in the hepatocyte contents of the positive effectors fructose 1.6-bisphosphate and fructose 1-phosphate were found at all concentrations of fructose up to 10mM. 4. Removal of the extrahepatocyte medium from the hepatocytes by washing resulted in an activation of the enzyme at all concentrations of fructose examined. 5. Inhibitors of the enzyme were shown to accumulate in the hepatocytes despite the depletion of ATP (a known negative effector) caused by higher concentrations of fructose. Indeed the inhibition of pyruvate kinase appeared to be correlated to the depletion of ATP. 6. Alanine (a known inhibitor) was shown to accumulate in hepatocytes as a consequence of incubation with fructose. 7. Allantoin and uric acid were shown to be inhibitors of a partially purified pyruvate kinase preparation assayed both in the presence and in the absence of fructose 1.6-bisphosphate. 8. Allantoin, but not uric acid, accumulated in the extrahepatocyte medium as a result of incubation of the cells with 10 mM-fructose.     

Alanine Prevents the Reduction of Pyruvate Kinase Activity in Brain Cortex of Rats Subjected to Chemically Induced Hyperphenylalaninemia

The mechanisms by which phenylalanine is toxic to the brain in phenylketonuria are not fully understood. Considering that brain glucose metabolism is reduced in these patients, our main objective was to determine pyruvate kinase activity in brain cortex of rats subjected to acute and chronic chemically induced hyperphenylalaninemia. The effect of alanine administration on the enzyme activity in the treated rats was also investigated. We also studied the in vitro effect of the two amino acids on pyruvate kinase activity in brain cortex of nontreated rats. The results indicated that phenylalanine inhibits pyruvate kinase in vitro and in vivo and that alanine prevents the inhibitory effect of phenylalanine on the enzyme activity. Considering the crucial role pyruvate kinase plays in glucose metabolism in brain, it is possible that inhibition of this enzyme activity may contribute to the brain damage characteristic of this disease.     

Pyruvate Kinase Activity in Crude Extracts of Leaves of Cynodon dactylonand Other C4Plants

A new extraction procedure and an LDH-coupled assay method are presented for the study of pyruvate kinase (PK) in leaf crude extracts from Cynodon dactylon(L.) Pers and other C₄plants. Extraction at pH 6.8 and assay at pH 6.2 facilitated the measuring of PK activity by eliminating phosphoenolpyruvate carboxylase interference more effectively than the thermal inactivation or chemical inhibition previously used. The method suggested did not affect the kinetic properties of PK as compared to the purified enzyme from C. dactylon.     

Modification of pyruvate kinase activity by proteins from chicken liver

The partial purification of a protein fraction inhibiting pyruvate kinase isoenzymes is described. The fraction was isolated from the (NH4)2SO4 step of the purification procedure for pyruvate kinase isoenzymes from chicken liver (Eigenbrodt, E. & Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1033-1046) by extraction with 1N NaOH, acidification to pH 3, ethanol precipitation and chromatography of the supernatant on DEAE-cellulose. The inhibitor fraction was further purified by disc gel electrophoresis using a gel gradient from 10 to 25%; this procedure separated activating proteins from the inhibitor fraction. The inhibitor fraction inhibited the pyruvate kinase isoenzymes from chicken in the sequence of decreasing effect: M2 greater than L greater than M1. The inhibition was due to a decrease in the affinity for phosphoenolpyruvate. The inhibitor is stable against heating for 5 min in 1% sodium dodecyl sulfate at 100 degrees C; it is destroyed by pepsin digestion. The inhibitor fraction could be purified further only by dodecyl sulfate gel electrophoresis. This resulted in the separation of 2 inhibitors (Mr = 33,500 +/- 8500 and ca. 5000), an activator (Mr = 15,100 +/- 5200), and an unidentified protein (Mr = 27,000).     

Comparative study of free and bound glycolytic enzymes from sea scorpion brain.

Free and bound forms of hexokinase, pyruvate kinase, and lactate dehydrogenase were prepared from the brain of the sea scorpion (Scorpaena porcus) in a low ionic strength medium. Properties of the free and bound forms were compared to determine whether binding to particulate matter could influence enzyme function or stability in vivo. Changes in pH differently affected the activity of the free and bound forms of all three enzymes. Furthermore, bound forms of hexokinase and pyruvate kinase were more stable than the free enzymes to heating at 45 degrees C. Bound hexokinase showed higher affinity for substrates (ATP, glucose) than the free form and bound lactate dehydrogenase had greater affinity for pyruvate and NADH. Although the affinities of the two forms of pyruvate kinase for substrates were similar, Hill coefficients for phosphoenolpyruvate as well as inhibition by ATP differed between the two enzyme forms. Free and bound lactate dehydrogenase also showed differences in Hill coefficients and bound lactate dehydrogenase was less sensitive to substrate inhibition by high pyruvate concentrations. The possible physiological role of the binding of these glycolytic enzymes to subcellular structures is discussed.     

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.