Phosphoenolpyruvate Binding Research Articles

Studies on the mechanism and stereochemical properties of the oxalacetate decarboxylase activity of pyruvate kinase.

When cod fish muscle oxalacetate decarboxylase catalyzes the decarboxylation of oxalacetate in the presence of NaBH4, L-lactate results from the reduction of enzyme-bound pyruvate. However, D-lactate results when borohydride reduces the binary enzyme-pyruvate complex formed by adding pyruvate from solution, as reported by others. This observation suggests that there are alternate mechanisms for reduction that are either kinetically or sterically determined for the E-pyruvate forms produced in the two directions. In the process of investigating the mechanism of reduction, the cod fish muscle decarboxylase was discovered to be identical with pyruvate kinase. Decarboxylase activity appears to take place at a site which overlaps the phosphoenolpyruvate binding site on this enzyme, as discussed in the following paper. Crystalline rabbit muscle pyruvate kinase also contains significant decarboxylase activity indicating that the two reactions may be structurally related functions. In the presence of K+, orthophosphate, or ATP the rabbit muscle enzyme catalyzes the detritiation of enzyme-bound pyruvate formed during decarboxylation before release of pyruvate from the enzyme, in analogy with the detritiation of pyruvate formed from P-[3-3/]enolpyruvate in the kinase reaction. This observation is consistent with the formation of an enolpyruvate intermediate common to the kinetic pathways of both reactions. Since the decarboxylase reac.tion is completely stereospecific, within the limits of detection, going with retention of configuration, the protonation of the enolpyruvate intermediate is completely determined by the enzyme as is the case with the enolpyruvate intermediate generated from P-enolpyruvate in the kinase reaction.

Anacystis nidulans mutants resistant to aromatic amino acid analogues

Three classes of mutants of Anacystis nidulans were selected on the basis of resistance to fluorophenylalanine and 2-amino-3-phenylbutanoic acid. The most frequent type exhibited DAHP synthetase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate-D-erythrose-4-phosphate-lyase [pyruvate phosphorylating], EC 4.1.2.15) activity identical to that of the parental strain. The second type was characterized by extremely low levels of the activity. The third type had a DAHP synthetase showing decreased sensitivity to inhibition by L-tyrosine. The enzyme was purified 140-fold from wild-type and feedback-insensitive strains, and the kinetics of the reaction was examined. The activity of the wild-type enzyme was inhibited 75% in the presence of 2.0 X 10-3 M tyrosine, and the altered enzyme was inhibited 10%. The following apparent constants were obtained from kinetic studies with partially purified wild-type enzyme: S0.5 for D-erythrose-4-phophate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 1.4 X 10-4 M. Inhibition by tyrosine was mixed with respect to binding of both D-erythrose-4-phosphate and phosphoenolpyruvate. In addition, tyrosine promoted cooperative interactions in the binding of phosphoenolpyruvate. For the altered enzyme the following apparent constants were obtained: S0.5 for D-erythrose-4-phosphate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 2.9 X 10-4 M. Inhibition by tyrosine was mixed with respect to D-erythrose-4-phosphate and competitive with respect to phosphoenolpyruvate. Tyrosine did not promote cooperative effects in the binding of phosphoenolpyruvate to the altered enzyme.

Bovine Pyruvate Kinases: I. PURIFICATION AND CHARACTERIZATION OF THE SKELETAL MUSCLE ISOZYME

Abstract Pyruvate kinase has been purified from bovine skeletal muscle by a procedure that includes only heat, ammonium sulfate fractionation, and chromatography on carboxymethyl Sephadex. Crystallization was accomplished by dialysis at 4° against 47% saturated ammonium sulfate. Since this simple purification scheme, with only slight modifications, has also been used to isolate turkey and rabbit muscle pyruvate kinases, it may be generally applicable to the isolation of muscle pyruvate kinase from birds and mammals. Bovine muscle pyruvate kinase prepared by the procedure described here was found to be homogeneous, as determined by disc gel electrophoresis at pH 9.5, gel electrophoresis in the presence of sodium dodecyl sulfate, sedimentation velocity and sedimentation equilibrium, isoelectric focusing, and immunodiffusion. It sediments at 9.9 S (s020,w) and has a maximum velocity of 400 µmoles per min per mg or more at 25°, or 2.4 times that value at 37°. The enzyme has maximal activity at pH 7.1, with an apparent Michaelis constant for phosphoenolpyruvate of 0.04 mm or less, and for ADP of 0.4 mm. Its isoelectric pH during electrofocusing is 8.9. Sedimentation equilibrium yielded a molecular weight of 230,000 with a range of ±3,000 in dilute phosphate buffer, and 57,000 ± 1,500 in 3.5 m guanidine hydrochloride, using a partial specific volume of 0.740 ml per g calculated from the amino acid composition. The molecular weight in guanidine hydrochloride, confirmed by gel electrophoresis in sodium dodecyl sulfate, indicates the presence of four polypeptide chains in the native enzyme. Binding studies suggest a total of four phosphoenolpyruvate binding sites, or one per subunit. Ion sensitivities and other chemical and physical parameters are very similar to those reported for rabbit muscle pyruvate kinase.

Gadolinium as a probe of the alkaline earth and ATP-metal binding sites in pyruvate kinase

The rare earth gadolinium forms a binary enzyme-metal complex with muscle pyruvate kinase which enhances the water proton relaxation rate ( ϵ b = 12 ± 2). Analysis of a Scatchard plot of the binding data indicates 3.7 ± 0.5 gadolinium binding sites with K d = 26 ± 10 μM per protein of 237,000 daltons. The transition metal ion, manganese, is displaced from the enzyme by the rare earths, gadolinium, neodymium, thulium, and lanthanum as well as the alkaline earths, magnesium and calcium suggesting all of these metal ions bind to the same site on the protein. Upon addition of ATP to a solution of gadolinium and enzyme a decrease in enhancement is observed which is consistent with the formation of a metal bridge complex. Because of the low dissociation constant for the Gd-ATP complex (0.1 μ m) it is possible to directly measure the dissociation of the Gd-ATP complex from the ternary enzyme-Gd-ATP complex, K 2 = 13 μM ± 4 μM. However, a ternary complex of phospho-enolpyruvate-Gd-enzyme is not detected by water proton relaxation rate enhancement measurements which leads to speculation that the ionic radius of gadolinium (0.94 Å) is so large that it results in a distortion of the phosphoenolypyruvate binding site on pyruvate kinase thus preventing phosphoenolpyruvate binding.

Thallium-205 Nuclear Magnetic Resonance Study of Pyruvate Kinase and Its Substrates: EVIDENCE FOR A SUBSTRATE-INDUCED CONFORMATIONAL CHANGE

Abstract A thallium-205 nuclear magnetic resonance (NMR) study of pyruvate kinase has been carried out. Shift and broadening of the 205T1 resonance have been observed upon binding of the thallous ion to the enzyme. Small changes in these parameters are observed upon binding of magnesium(II) to the enzyme with further changes arising from the binding of phosphoenolpyruvate, a substrate of the enzyme. The longitudinal (T1) and transverse (T2) relaxation times of 205T1 are dramatically affected by the presence of the paramagnetic manganous ion due to the existence of electronnuclear dipolar (for T1 and T2) and hyperfine (for T2 only) interactions. The distance between the monovalent (T1+) and divalent (Mn2+) activators of pyruvate kinase has been evaluated from the T1 values. In the pyruvate kinase-Mn(II) complex the average distance is 8.2 A whereas in the pyruvate kinase-Mn(II)-phosphoenolpyruvate it is 4.9 A. The change of the thallium to manganese distance upon binding of phosphoenolpyruvate is direct evidence for a substrate-induced conformational change. The thallous ion forms complexes with the substrates of pyruvate kinase (phosphoenolpyruvate, ATP, ADP, and pyruvate) in absence of the enzyme, which give rise to chemical shifts and broadenings of the 205T1 resonance. The line broadening is enhanced in presence of Mn(II) suggesting that the substrates form complexes having both a monovalent and a divalent ion. The possibilities of using the NMR properties of the nuclei of the alkali metal ions to probe macromolecular environment in the presence of paramagnetic ions are discussed. It appears that the properties of thallium-205 are uniquely suitable in this regard.

Conformational Differences in the Active Sites of Muscle and Erythrocyte Pyruvate Kinase

Abstract We have examined the inhibition of the allosteric pyruvic kinase isozyme of human erythrocytes (PK I) and of the nonallosteric pyruvic kinase isozyme of rabbit muscle (PK III) by iodoacetamide and the protection against this inhibition by substrates and cations. Both isozymes are inhibited in a similar fashion which can be accounted for by alkylation of two different groups on each isozyme, one with a pKa of less than 6.8 and the other with a pKa of 7.8. Under the experimental conditions used, the effects observed in the protection studies were predominantly those involving the group with a pKa below 6.8. The rabbit muscle enzyme is not protected from iodoacetamide inhibition by phosphoenolpyruvate, ADP, or magnesium alone. It is protected by potassium or the magnesium-ADP complex. At pH 7.4, the human erythrocyte enzyme is not protected by phosphoenolpyruvate, ADP, or magnesium alone, or by potassium or the magnesium-ADP complex. In the presence of either of the positive allosteric effectors of the erythrocyte enzyme, phosphoenolpyruvate or fructose 1,6-diphosphate, both potassium and the magnesium-ADP complex have a protective action. We conclude that PK I exists in two conformational states, one equivalent to the T form of the Monod, Wyman, and Changeaux, model (Monod, J., Wyman, J., and Changeaux, G. P., J. Mol. Biol., 12, 88 (1965)) with a low affinity for phosphoenolpyruvate and no affinity for potassium or the magnesium-ADP complex, and the other equivalent to the R form of this model with a high affinity for phosphoenolpyruvate, potassium, and magnesium-ADP. Binding of either phosphoenolpyruvate or fructose 1,6-diphosphate converts the T form into the R form. PK III always exists in the R form with a high affinity for phosphoenolpyruvate, potassium, and the magnesium-ADP complex. We have demonstrated a cooperative influence of phosphoenolpyruvate on potassium binding to the human erythrocyte enzyme which is consistent with this allosteric model.

Tyrosine-inhibited 3-deoxy- d- arabino-heptulosonate 7-phosphate synthetase : Properties of the partially purified enzyme from Salmonella typhimurium

Tyrosine-regulated 3-deoxy- d- arabino-heptulosonate 7-phosphate (DAHP) synthetase has been partially purified from Salmonella typhimurium. Properties of the enzyme and aspects of the reaction mechanism were studied. Lineweaver-Burk plots for saturation by phosphoenolpyruvate and erythrose 4-P yield families of parallel or nearly parallel lines suggesting the possibility of a ping-pong mechanism. A sequential mechanism is, however, not excluded. Stabilization of the enzyme to heat inactivation by phosphoenolpyruvate suggests that erythrose 4-P is not required for binding of phosphoenolpyruvate. The DAHP synthetase reaction was studied in ( 18O)H 2O. P i, formed concomitantly with synthesis of DAHP, does not contain 18O. This requires splitting of the CO bond of phosphoenolpyruvate and indicates direct elimination of phosphate. These results suggest that the enzyme reacts with phosphoenolpyruvate to form an uncharacterized enzyme-substrate intermediate and P i. Reaction of erythrose 4-P with the enzyme-substrate intermediate could yield the product DAHP and enzyme. Feedback inhibition of the enzyme by tyrosine is dependent upon pH. The Hill coefficient, n′, is 1.4 at both pH 6.0 (85% inhibition by 0.1 m m tyrosine) and at pH 8.0 (25% inhibition by 0.1 m m tyrosine), thus suggesting cooperative interaction of tyrosine sites. Treatment of the enzyme with p-mercuribenzoate causes activation at concentrations lower than 1 μ m and inhibition at higher concentrations but does not affect inhibition by tyrosine. Enzymatic activity is dependent on Co 2+.

Mechanisms of fructose diphosphate activation of a mutant pyruvate kinase from human red cells

1. 1. Normal human erythrocyte pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) and mutant pyruvate kinase from a propositus with congenital, nonspherocytic, hemolytic anemia were isolated by DEAE-cellulose extraction and (NH 4) 2SO 4 precipitation. 2. 2. Fru-1,6-P 2 lowered the apparent Michaelis constant for phosphoenolpyruvate of the normal enzyme but did not substantially change the maximal velocity. This activator greatly elevated both the apparent Michaelis constant for phosphoenolpyruvate and the maximalvelocity of the mutant enzyme. 3. 3. Fru-1,6-P 2 did not affect the Michaelis constant for ADP or the optimal Mg 2+ concentration in either the normal or the mutant enzyme. 4. 4. Fru-1,6-P 2 altered the extremely sensitive interaction of the binding sites for the two substrates on the mutant enzyme. 5. 5. The sensitivity of the normal enzyme to Fru-1,6-P 2 could be increased by either dilution or heating. With heating, dilute solutions of both normal and mutant enzymes decayed in two steps, indicating the presence of multiple conformations in both enzymes. 6. 6. A molecular model is proposed for Fru-1,6-P 2 activation; Fru-1,6-P 2 acts directly on the phosphoenolpyruvate binding site; interaction of the two substrate sites then alters the conformation of the binding site for ADP.

The Enzymatic Carboxylation of Phosphoenolpyruvate: I. PURIFICATION AND PROPERTIES OF PHOSPHOENOLPYRUVATE CARBOXYLASE

Abstract Phosphoenolpyruvate carboxylase has been isolated from germinating peanut cotyledons and purified 2700-fold. The purified enzyme is approximately 80% pure as judged from sedimentation patterns, has a sedimentation coefficient (s20,w) of 13.9 S, and catalyzes the carboxylation of 50 µmoles of phosphoenolpyruvate per min per mg of protein (refractometrically determined) at 30°. Enzymatic carboxylation occurs optimally at pH 8.0 to 8.2. The Km values determined for HCO3-, phosphoenolpyruvate, and Mg2+ are 3.1 x 10-4, 5 to 6 x 10-4, and 3 to 4 x 10-4 m, respectively, at pH 7.9. Studies on the effect of pH on the Km values for phosphoenolpyruvate and Mg2+ were conducted. Evaluation of plots of -log Km (Mg2+ and phosphoenolpyruvate) against pH indicate that a dissociable group (or groups) of the carboxylase having a pK of 7.3, presumably an imidazole group, is involved in Mg2+ and phosphoenolpyruvate binding. Other evidence supporting this view is presented. During the enzymatic carboxylation of phosphoenolpyruvate, 18O from substrate HC18O3- is incorporated into the orthophosphate and oxalacetate reaction products in a ratio of 1:2. This indicates that the HCO3- (or CO32-) anion rather than CO2 is the active species in the carboxylation reaction. A mechanism is proposed which is consistent with this and other observations on the phosphoenolpyruvate carboxylase-catalyzed reaction.

Kinetic and Magnetic Resonance Studies of the Pyruvate Kinase Reaction: II. COMPLEXES OF ENZYME, METAL, AND SUBSTRATES

Abstract A correlation between the kinetics of the pyruvate kinase reaction and the enhancements of the relaxation rate of the nuclear spins of water protons in the ternary enzyme-manganese-substrate (E-Mn-S) complexes, et, has revealed certain salient features of the pathway of the reaction and the structure of the ternary intermediates. The rate equation for initial velocities of the forward reaction was derived by assuming (a) rapid simultaneous equilibria of enzyme with divalent metal ion, adenosine diphosphate, manganese adenosine diphosphate, and phosphoenolpyruvate, (b) random order of combination, and (c) phosphoryl transfer as the rate-determining step. This formulation permitted the calculation of the dissociation constants Ks of the ES complexes and K3 of the EMS complexes for ADP and phosphoenolpyruvate. The values of K3 for E-Mn-ADP and E-Mn-phosphoenolpyruvate were determined directly by equilibrium experiments with proton relaxation rate (PRR) with the use of et as the parameter which characterized the ternary complexes. The values of Ks for E-phosphoenolpyruvate and E-ADP were determined directly by equilibrium experiments with the use of the kinetic protection method with p-chloromercuribenzoate inactivation. The agreement of the dissociation constants of the binary and ternary enzyme complexes determined by equilibrium methods with the kinetically calculated ones, taken in conjunction with the previously established agreement of the dissociation constant of EM with its kinetically calculated activator constant, validates the assumed random binding of metal, ADP, MnADP, and phosphoenolpyruvate to the enzyme. The inhibitor constant, Ki, for ATP agreed well with its K3 determined by PRR. In the ternary pyruvate complex, the results from PRR data differed from the kinetic data in two ways: pyruvate was not competitive with phosphoenol pyruvate, and K3 from PRR data was an order of magnitude lower than Ki obtained kinetically. The value of K3 was raised to the Ki value when the PRR titration was performed in the presence of ADP or inorganic phosphate, suggesting that the breakdown of the product complex in the pyruvate kinase reaction proceeds by a preferred order in which pyruvate dissociates first. The PRR enhancement values of all the ternary EMS complexes, et, were found to be less than eb, the enhancement of the binary complex; this decrease may be ascribed in part to the replacement of water ligands by groups on the substrate. The magnitude of the decrease of et(ATP) with respect to et(ADP) is consistent with a metal bridge structure in which the divalent cation is coordinated both to the enzyme and to the γ-phosphoryl group of ATP in the active complex. No data have been found which contravene a metal bridge structure. Kinetic data which favor this structure may be adduced from the relative binding constants of substrates to the manganese- and magnesium-activated enzyme; changing the divalent activator from manganese to magnesium, which has a lower affinity for ligands, raised the K3 of phosphoenolpyruvate and the Ki of ATP and of pyruvate by factors of 2.6, 5.1, and 2.6, respectively, but it had little effect on the K3 of ADP. The structure of the E-Mn-phosphoenolpyruvate complex as reflected in the finding that et(phosphoenolpyruvate) << eb requires a change in the environment of the manganese other than mere replacement of water ligands; such a change may be ascribed to a conformational change in the protein at the binding site.