Electron paramagnetic resonance and water proton relaxation rate studies of formyltetrahydrofolate synthetase-manganous ion complexes. Evidence for involvement of substrates in the promotion of a catalytically competent active site.

Conformational properties of the active site of formyltetrahydrofolate synthetase from Clostridium cylindrosorum have been examined by EPR spectroscopy and by solvent proton relaxation rate (PPR) studies of manganous complexes with the enzyme. Ternary enzyme-Mn-nucleotide complexes give EPR spectra which are very similar to those for the binary Mn-nucleotide complexes. However, upon addition of tetrahydrofolate to form the quaternary complexes, enzyme-MnADP-tetrahydrofolate and enzyme MnATP-tetrahydrofolate the EPR line shapes are changed substantially. Spectra for the quaternary complexes exhibit narrow line widths, and the splitting patterns are characteristic of a slightly asymmetric electronic environment for the bound Mn(II). Addition of formate to the ADP quatenary complex induces a further significant narrowing of the EPR line widths, although in the absence of tetrahydrofolate, formate does not influence the EPR spectrum for the enzyme-MnADP species. Both Pi and nitrate cause changes in the EPR patterns for the higher complexes of the enzyme which involve both ADP and tetrahydololate. However, the Pi effect is not influenced by the presence of formate whereas the characteristic effect of nitrate is potentiated only when formate is present. EPR sectra for the thernary complex with the beta, gamma-methylene analog of ATP App(CH2)p differ significantly from spectra for the binary App(CH)p complex is not influenced by further additions of tetrahydrofolate and of tetrahydorfolate and formate. The failure of spectra for the App(CH)p complex to respond to additions of the other substrates for the reaction is in marked contrast to the behavior found for the natural nucleotide substrates and is tentatively attributed to the lack of a protein-mediated interaction between the nucleotide and tetrahydrofolate binding sites in the analog complex. The frequency dependence of solvent PRR in the presence of the various complexes allows an estimate of the correlation times for electron-nuclear dipolar interaction and thereby the extent of hydration of the bound Mn(II) among the various complexes..     

7Li, 31P, and 1H NMR studies of interactions between ATP, monovalent cations, and divalent cation sites on rabbit muscle pyruvate kinase

The interactions between ATP, monovalent cations, and divalent cations on rabbit muscle pyruvate kinase have been examined using 7Li, 31P, and 1H nuclear magnetic resonance. Water proton nuclear relaxation studies are consistent with the binding of Li+ to the K+ site on pyruvate kinase with an affinity of 120 mM in the absence of substrates and 16 mM in the presence of P-enolpyruvate. Titrations with pyruvate demonstrate that pyruvate binds to the enzyme with an affinity of 0.65 mM in the presence of Li+ and 0.4 mM in the presence of K+. 7Li+ nuclear relaxation rates in solutions of pyruvate kinase are increased upon titration with the metal-nucleotide analogue, Cr(H2O)4ATP. Mn2+ EPR spectra were used to determined the distribution of the enzyme between the so-called isotropic and anisotropic conformations of the enzyme (Ash, D. E., Kayne, F., and Reed, G.H. Arch. Biochem. Biophys. (1978) 190, 571-577). Li-Cr distances of 5.6 and 11.0 A were calculated for the anisotropic and isotropic forms, respectively, in the absence or presence of pyruvate. When the divalent cation site on the enzyme was saturated with Mg2+, these distances increased to 6.7 and 9.5 A, respectively, regardless of the presence or absence of pyruvate. 31P nuclear relaxation studies with the diamagnetic metal-nucleotide analogue, Co(NH3)4ATP, indicated that addition of Mn2+ ion to the divalent cation site on the enzyme increased the longitudinal relaxation rates of all three phosphorus nuclei of the analogue. The 31P data indicate that the presence of pyruvate at the active site effects a decrease in the Mn-P distances, bringing Mn2+ and Co(NH3)4ATP closer together at the active site. The data also permit an evaluation of the role of the metal coordinated to the beta-P and gamma-P of ATP at the active site.     

Equilibrium and water proton relaxation rate enhancement properties of formyltetrahydrofolate synthetase-manganous ion-substrate complexes.

Binding of Mn(pi)-nucleotide complexes to the enzyme formyltertrahydrofolate synthetase (EC 6.3.4.3) from Clostridium cylindrosporum has been examined in the presence and absence of other substrates by solvent proton relaxation mearurements. MnADP and MnATP form ternary complexes with the enzyme with highly enhanced proton relaxation rates for water. The enhancement parameters, epsilont, for the MnADP and MnATP ternary complexes are 19.8 and 12.5, respectively at 24.3 MHZ and 25 degrees. Titration curves with constant total concentrations of enzyme and Mn(pi) with variable nucleotide concentration are similar to those observed in similar titrations with the endp and MnATP are 175 muM and 64 muM, respectively at 25 degrees. Addition of tetrahydrofolate to solutions of the MnADP OR MnATP ternary complexes lowers the observed relaxation enhancement markedly. An analysis of titration curves with constant total concentrations of enzyme, Mn(pi), and nucleotide with variable tetrahydrofolate concentration gives the dissociation constant for tetrahydrofolate from the respective quaternary complexes. The affinity of the enzyme for tetrahydrofolate is increased 6-fold when MnADP is present at the active site whereas a 3-fold increase is observed with MnATP present. Furthermore, there is a 20-fold increase in the enzyme's affinity for tetrahydrofolate when both MnADP and the third substrate, formate, are present. The observed relaxation rate of water for solutions of the complex, enzyme-MnADP-tetrahydrofolate-formate, is deenhanced with respect to the rate observed for the simple aquo-Mn(pi) solution. Addition of nitrate to solutions of the above complex increases the affinity of the enzyme for tetrahydrofolate and MnADP by an additional factor of 5 and lowers the relaxation rate further to a value which approaches that for solutions of the enzyme and substrates which lack the paramagnetic cation.     

Structural characterization of manganese(II)-nucleotide complexes bound to yeast 3-phosphoglycerate kinase: 13C relaxation measurements using [U-13C]ATP and [U-13C]ADP

A complete characterization of the conformations of Mn.ADP and Mn.ATP bound to the active site of yeast 3-P-glycerate kinase is presented. These conformations have been deduced on the basis of paramagnetic effects on 13C spin-lattice relaxation rates in [U-13C]nucleotides due to Mn(II), used as a substituent activating cation. The 13C relaxation measurements were performed on exclusively enzyme-bound complexes E.Mn.[U-13C]ATP and E.Mn.[U-13C]ADP at three distinct 13C NMR frequencies: 75.4, 125.7, and 181 MHz. The frequency dependence of the relaxation data has been analyzed in an effort to evaluate distances from the cation for all 10 13C nuclei in the adenosine moieties of E.Mn.ATP and E.Mn.ADP. These distance data, taken along with previously published cation-31P distances, have been used as constraints in the molecular modeling program Quanta, in which molecular dynamics simulations and energy minimization have been performed to determine the conformations that are compatible with the distance data. It was possible to model the distances on the basis of a single enzyme-bound conformation for each of the nucleotides. The details of the enzyme-bound Mn.ATP and Mn.ADP conformations are distinguishably different from each other, indicating that structural alterations occur in the enzyme-bound reaction complex as the enzyme turns over. For example, when the adenosine moieties in the bound structures of Mn.ATP and Mn.ADP are superposed, the cation is found to be displaced by approximately 2.4 A between the two conformations, suggesting that these structural changes may involve movements with significant amplitudes. Furthermore, the NMR-determined structures of enzyme-bound Mn.ATP and Mn.ADP are significantly different from those in published X-ray crystal structures of the enzyme-nucleotide complexes.     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

3-Mercaptopicolinate. A reversible active site inhibitor of avian liver phosphoenolpyruvate carboxykinase

The inhibition of chicken liver phosphoenolpyruvate carboxykinase by 3-mercaptopicolinic acid (3-MP) has been investigated. Kinetic studies show 3-MP to be a noncompetitive inhibitor relative to all substrates and to the activator, Mn2+. EPR studies demonstrate that Mn2+ binding to the enzyme is unaffected by 3-MP. Proton relaxation rate studies demonstrate that 3-MP binds to the binary E X Mn complex with a KD of 0.5 X 10(-6) M and gives a ternary enhancement of 8.0. Additional proton relaxation rate studies detected formation of the quaternary complexes E X Mn X IDP X 3-MP, E X Mn X ITP X 3-MP, and E X Mn X CO2 X 3-MP. High resolution 1H nuclear relaxation rate studies suggest that 3-MP binds in close proximity to the activator cation, Mn2+, but not in the first coordination sphere. Active site models suggest that the 3-MP-binding site may partially overlap the phosphoenolpyruvate-binding site. The NMR studies, which detected formation of the quaternary E X Mn X 3-MP X phosphoenolpyruvate complex, also demonstrated that the binding of one of these ligands affects the interactions of the other ligand with E X Mn. Calorimetric studies of the E X Mn complex demonstrated that 3-MP causes an increase in the transition temperature midpoint without an increase in enthalpy. These results indicate that 3-MP causes a conformational change in the enzyme but does not increase the thermostability of the ternary complex. The experiments reported herein suggest that inhibition by 3-MP is due to specific and reversible binding within the active site of phosphoenolpyruvate carboxykinase.     

A nuclear magnetic resonance study of the topography of binding sites of Escherichia coli carbamoyl-phosphate synthetase

Two paramagnetic probes, viz., Mn2+ and Cr3+-ATP, were used to map distances to various loci on carbamoyl-phosphate synthetase by using NMR measurements. The paramagnetic influence of Mn2+ on the 1H of L-glutamate and L-ornithine was measured at 200 and 360 MHz. On the basis of these data, a correlation time for the paramagnetic interaction was determined (2 X 10(-9) s) and used to compute distances. These were in the range 7-9 A. Distances were also calculated from Mn2+ to the 13C-5 atom of glutamate (8.6 A), to the monovalent cation site (approximately 8 A), and to the phosphorus atoms of ATP in the Co(NH3)4ATP complex. For studies of the monovalent cation site relaxation rates of 6Li+, 7Li+, and 15NH4+ were measured. With Cr3+ ATP as a paramagnetic substrate analogue, Cr3+ to 13C distances were measured with the substrates HCO3(-) and [5-13C]glutamate. These NMR data provide the first topographical map of the arrangement of substrates, metal ion activators, and allosteric modifiers on the Escherichia coli carbamoyl-phosphate synthetase dimer.     

Activation and inhibition of rabbit muscle pyruvate kinase by transition-metal ions.

The paper reports a comparative study of the effects of Mn2+, Ni2+ and Co2+ on the reaction of ADP with phosphoenolypyruvate when catalysed by K+-activated rabbit muscle pyruvate kinase. The activation and subsequent inhibition that occurs as the bivalent ion concentration is increased is taken as evidence that the substrates of the enzyme are phosphoenolypyruvate, uncomplexed ADP and free bivalent cation. Kinetic constants for the binding of the bivalent cation to the enzyme are reported.     

Ketopantoate hydroxymethyltransferase. II. Physical, catalytic, and regulatory properties.

Some physical, catalytic, and regulatory properties of ketopantoate hydroxymethyltransferase (5,10-methylenetetrahydrofolate: alpha-ketoisovalerate hydroxymethyltranferase) from Escherichia coli are described. This enzyme catalyzes the reversible synthesis of ketopantoate (Reaction 1), an essential precursor of pantothenic acid. (1) HC(CH3)2COCOO- + 5,10-methylene tetrahydrofolate f in equilibrium r HOCH2C(CH3)2COCOO- + tetrahydrofolate It has a molecular weight by sedimentation equilibrium of 255,000, a sedimentation coefficient (S20,w) of 11 S, a partial specific volume of 0.74 ml/g, an isoelectric point of 4.4, and an absorbance, (see article), of 0.85. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and amino acid analyses give a subunit molecular weight of 27,000 and 25,700, respectively; both procedures indicate the presence of 10 identical subunits. The NH2-terminal sequence is Met-Tyr---. The enzyme is stable and active over a broad pH range, with an optimum from 7.0 to 7.6. It requires Mg2+ for activity; Mn2+, Co2+, Zn2+ are progressively less active. The enzyme is not inactivated by borohydride reduction in the presence of excess substrates, i.e. it is a Class II aldolase. Reaction 1f is partially inhibited by concentrations of formaldehyde (0.8 mM) and tetrahydrofolate (0.38 mM) below or near the Km values, apparent Km values are 0.18, 1.1 and 5.9 mM for tetrahydrofolate, alpha-ketoisovalerate, and formaldehyde, respectively. For Reaction 1r, apparent Km values are 0.16 and 0.18 mM, respectively, for ketopantoate and tetrahydrofolate, and the saturation curves for both substrates show positive cooperativity. Forward and reverse reactions occur at similar maximum velocities (Vmax approximately equal to 8 mumol of ketopantoate formed or decomposed per min per mg of enzyme at 37 degrees). Only 1-tetrahydrofolate is active in Reaction 1; d-tetrahydrofolate, folate, and methotrexate were neither active nor inhibitory. However, 1-tetrahydrofolate was effectively replaced with conjugates containing 1 to 6 additional glutamate residues; of these, tetrahydropterolpenta-, tetra-, and triglutamate were effective at lower concentrations than tetrahydrofolate itself; they were also the predominant conjugates of tetrahydrofolate present in E. coli. Alpha-Ketobutyrate, alpha-ketovalerate, and alpha-keto-beta-methylvalerate replaced alpha-ketoisovalerate as substrates; pyruvate was inactive as a substrate, but like isovalerate, 3-methyl-2-butanone and D- or L-valine, inhibited Reaction 1. the transferase has regulatory properties expected of an enzyme catalyzing the first committed step in a biosynthetic pathway. Pantoate (greater than or equal to 500 muM) and coenzyme A (above 1 mM) all inhibit; the Vmax is decreased, Km is increased, and the cooperativity for substrate (ketopantoate) is enhanced. Catalytic activity of the transferase is thus regulated by the products of the reaction path of which it is one component; transferase synthesis is not repressed by growth in the presence of pantothenate.     

Role of the divalent metal cation in the pyruvate oxidase reaction

Purified pyruvate oxidase requires a divalent metal cation for enzymatic activity. The function of the divalent metal cation was studied for unactivated, dodecyl sulfate-activated, and phosphatidylglycerol-activated oxidase. Assays performed in the presence of Mg2+, CA2+, Zn2+, Mn2+, Ba2+, Ni2+, Co2+, Cu2+, and Cr3+ in each of four different buffers, phosphate, 1,4-piperazinediethanesulfonic acid, imidazole, and citrate, indicate that any of these metal cations will fulfill the pyruvate oxidase requirement. Extensive steady state kinetics data were obtained with both Mg2+ and Mn2+. All the data are consistent with the proposition that the only role of the metal is to bind to the cofactor thiamin pyrophosphate (TPP) and that it is the Me2+-TPP complex which is the true cofactor. Values of the Mg2+ and Mn2+ dissociation constants with TPP were determined by EPR spectroscopy and these data were used to calculate the Michaelis constant for the Me2+-TPP complexes. The results show that the Michaelis constants for the Me2+-TPP complexes are independent of the metal cation in the complex. Fluorescence quenching experiments show that the Michaelis constant is equal to the dissociation constant of the Mn2+-TPP complex with the enzyme. It was also shown that Mn2+ will only bind to the enzyme in the presence of TPP and that one Mn2+ binds per subunit. Steady state kinetics experiments with Mn2+ were more complicated than those obtained with Mg2+ because of the formation of an abortive Mn2+-pyruvate complex. Both EPR and steady state kinetics data indicated complex formation with a dissociation constant of about 70 mM.     

Mechanistic studies on E. coli DNA topoisomerase I: divalent ion effects.

E. coli DNA topoisomerase I catalyzes the hydrolysis of short, single stranded oligodeoxynucleotides. It also forms a covalent protein-DNA complex with negatively supercoiled DNA in the absence of Mg2+ but requires Mg2+ for the relaxation of negatively supercoiled DNA. In this paper we investigate the effects of various divalent metals on catalysis. For the relaxation reaction, maximum enzyme activity plateaus after 2.5 mM Mg2+. However, the rate of cleavage of short oligodeoxynucleotide increased linearly between 0 and 15 mM Mg2+. In the oligodeoxynucleotide cleavage reaction, Ca2+, Mn2+, Co2+, and Zn2+ inhibit enzymatic activity. When these metals are coincubated with Mg2+ at equimolar concentrations, the normal effect of Mg2+ is not detectable. Of these metals, only Ca2+ can be substituted for Mg2+ as a metal cofactor in the relaxation reaction. And when Mg2+ is coincubated with Mn2+, Co2+, or Zn2+ at equimolar concentrations, the normal effect of Mg2+ on relaxation is not detectable. We propose that Mg2+ allows the protein-DNA complex to assume a conformation necessary for strand passage and enhance the rate of enzyme turnover.     

Modifications of the adenylate cyclase complex during differentiation of cultured myoblasts

Alterations in receptor-independent activation of adenylate cyclase during proliferation and differentiation of L6E9 myoblasts were studied using Mn2+, forskolin, and Gpp(NH)p. Analyses were performed 3, 6, and 10 days following subculture, corresponding to onset of proliferation, end of proliferation with start of differentiation, and completion of differentiation, respectively. The apparent activation constant for Mn2+ decreases with the age of the culture; the apparent activation constant for Mg2+ does not. Bimodal activation by Mn2+, i.e., at concentrations greater than 10 mM, results in total adenylate cyclase activity less than the Vmax and occurs exclusively in differentiated cultures. Independent of the presence of Mg2+, forskolin activation occurs with low-and high-affinity constants in differentiated cultures and with a low affinity constant in youngest cultures; intermediate cultures (day 6) demonstrate low- and high-affinity activation only in the presence of high Mg2+. In contrast, the Vmax for forskolin increases with increasing Mg2+ in all culture ages. Although Gpp(NH)p-dependent adenylate cyclase activation occurs with an apparent activation constant independent of culture age and Mg2+, low Mg2+ fosters bimodal activation by Gpp(NH)p, i.e., above 100 microM nucleotide, total adenylate cyclase activity is less than the Vmax. The loss of stimulatory capacity by high Gpp(NH)p is greatest in differentiated cultures. Additional experiments are presented to substantiate that bimodal activation by Gpp(NH)p is specific. Cholera- and pertussis toxin-dependent ADP ribosylation patterns demonstrate a marked decrease in both Ns and Ni in differentiated cultures. The data suggest that alterations in postreceptor activation of adenylate cyclase during the course of differentiation and proliferation are mediated by guanine nucleotide binding proteins as well as by allosteric cation regulatory units.     

Phosphoenolpyruvate carboxykinase in mouse pancreatic islets. ATP-induced changes in sensitivity to Mn2+ activation

The presence of high phosphoenolpyruvate carboxykinase (EC 4.1.1.32) activity in mouse islet cytosol has been demonstrated. The enzyme was activated by Mn2+ with a Ka of 100 X 10(-6) mol/l. The mean total activity of the Mn2+-stimulated phosphoenolpyruvate carboxykinase in islet cytosol estimated at 22 degrees C with saturating concentrations of the substrates oxaloacetate and ITP was 146 pmol/min per micrograms DNA. Km was calculated to be 6 X 10(-6) mol/l for oxaloacetate and 140 X 10(-6) mol/l for ITP. The islet phosphoenolpyruvate carboxykinase activity was not increased after starvation of the animals for 48 h. Preincubation of the cytosol at 4 degrees C with Fe2+, quinolinate, ATP, Pi, glucose 6-phosphate, fructose 1,6-bisphosphate, NAD+, NADH, oxaloacetate, ITP, cyclic AMP and Ca2+ had no effect on the enzyme activity. However, preincubation of the cytosol at 37 degrees C with ATP-Mg inhibited the Mn2+-stimulated phosphoenolpyruvate carboxykinase activity progressively with time and in a concentration-dependent manner. A similar but weaker inhibitory effect was observed with p[NH]ppA, whereas p[CH2]ppA, ADP, AMP, adenosine and Pi had no effect. It is tentatively suggested that ATP and p[NH]ppA either by adenylation or otherwise affect the interaction between islet phosphoenolpyruvate carboxykinase and the recently discovered Mr = 29000 protein modulator of the enzyme in such a way - perhaps by causing a dissociation between them - that phosphoenolpyruvate carboxykinase loses its sensitivity to Mn2+ activation.     

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.     

1H and 31P nuclear magnetic resonance and kinetic studies of the active site structure of chloroplast CF1 ATP synthase

The interaction of nucleotides and nucleotide analogues and their metal complexes with Mn2+ bound to both the latent and dithiothreitol-activated CF1 ATP synthase has been examined by means of steady-state kinetics, water proton relaxation rate (PRR) measurements, and 1H and 31P nuclear relaxation measurements. Titration of both the latent and activated Mn(2+)-CF1 complexes with ATP, ADP, Pi, Co(NH3)4ATP, Co(NH3)4ADP, and Co(NH3)4AMPPCP leads to increases in the water relaxation enhancement, consistent with enhanced metal binding and a high ternary complex enhancement. Steady-state kinetic studies are consistent with competitive inhibition of CF1 by Co(NH3)4AMPPCP with respect to CaATP. The data are consistent with a Ki for Co(NH3)4AMPPCP of 650 microM, in good agreement with a previous Ki of 724 microM for Cr(H2O)4ATP [Frasch, W., & Selman, B. (1982) Biochemistry 21, 3636-3643], and a best fit KD of 209 microM from the water PRR measurements. 1H and 31P nuclear relaxation measurements in solutions of CF1 and Co(NH3)4AMPPCP were used to determine the conformation of the bound substrate analogue and the arrangement with respect to this structure of high- and low-affinity sites for Mn2+. The bound nucleotide analogue adopts a bent conformation, with the low-affinity Mn2+ site situated between the adenine and triphosphate moieties and the high-affinity metal site located on the far side of the triphosphate chain. The low-affinity metal forms a distorted inner-sphere complex with the beta-P and gamma-P of the substrate. The distances from Mn2+ to the triphosphate chain are too large for first coordination sphere complexes but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules or residues from the protein.     

Characterization of ammonium (methylammonium)/potassium antiport in Escherichia coli

The energetics of ammonium ion transport by Escherichia coli have been studied using [14C]methylammonium as a substrate. Rapid assays for uptake allowed kinetic parameters (CH3NH3+ Km = 36 microM; Vmax = 4 nmol X s-1 X mg-1 to be determined in the absence of CH3NH3+ metabolism. Cells cultured in media containing 1 mM NH4+ failed to express CH3NH3+ transport activity. Methylammonium accumulated at levels which were 100-fold higher than those of the medium. This accumulation was dependent upon the addition of glucose or pyruvate. The entry of CH3NH3+ supported by glucose oxidation in an F1F0-ATPase-deficient mutant was blocked by uncoupler. Transport by wild-type cells under similar conditions was significantly inhibited by arsenate. Thus, CH3NH3+ uptake requires both ATP and an electrochemical H+ gradient. This transport activity was lost upon exposure of E. coli to osmotic shock, but could be recovered by incubation of shocked cells with boiled shock fluid or with glucose plus K+ in the presence of chloramphenicol. Similar reconstitution was observed in K+-depleted parental strains, but not in a mutant defective in K+ transport, demonstrating a requirement for internal K+. However, external K+ proved to be a noncompetitive inhibitor (Ki = 1 mM) of CH3NH3+ uptake by K+ -replete bacteria. External Na+ had no effect on transport. The addition of NH4+ or CH3NH3+ induced a rapid exodus of intracellular 86Rb+, an analog which was able to substitute for K+. The molar ratio of CH3NH3+ uptake to Rb+ exit was 1.12 +/- 0.11. These findings support a mechanism for CH3NH3+ (NH4+) accumulation which requires K+ antiport (exchange) and is driven by the electrochemical K+ gradient.     

Metal ion activation of S-adenosylmethionine decarboxylase reflects cation charge density

S-Adenosylmethionine decarboxylase from Escherichia coli is a pyruvoyl cofactor-containing enzyme that requires a metal cation for activity. We have found that the enzyme is activated by cations of varying charge and ionic radius, such as Li+, A13+, Tb3+, and Eu3+, as well as the divalent cations Mg2+, Mn2+, and Ca2+. All of the activating cations provide kcat values within 30-fold of one another, showing that the charge of the cation does not greatly influence the rate-limiting step for decarboxylase turnover. Cation concentrations for half-maximal activation decrease by >100-fold with each increment of increase in the cation charge, ranging from approximately 300 mM with Li+ to approximately 2 microM with trivalent lanthanide ions. The cation affinity is related to the charge/radius ratio of the ion for those ions with exchangeable first coordination sphere ligands. The exchange-inert cation Co(NH3)63+ activates in the presence of excess EDTA (and NH4+ does not activate), indicating that direct metal coordination to the protein or substrate is not required for activation. The binding of metal ions (monitored by changes in the protein tryptophan fluorescence) and enzyme activation are both cooperative with Hill coefficients as large as 4, the active site stoichiometry of this (alphabeta)4 enzyme. The Hill coefficients for Mg2+ binding and activation increase from 1 to approximately 4 as the KCl concentration increases, which is also observed with NaCl or KNO3; neither Na+ nor K+ activates the enzyme. The single tryptophan in the protein is located 16 residues from the carboxyl terminus of the pyruvoyl-containing alpha chain, in a 70-residue segment that is not present in metal ion independent AdoMet decarboxylases from other organisms. The results are consistent with allosteric metal ion activation of the enzyme, congruent with the role of the putrescine activator of the mammalian AdoMet decarboxylase.     

酿酒酵母胞内无机焦磷酸酶的分离纯化及性质

An inorganic pyrophosphatase (EC3.6.1.1) from Saccharomyces cerevisiae was purified to PAGE homogeneity by sonication disruption. (NH4)2SO4 fractionation and DEAE-cellulose colunm chromatography. The optimum pH and temperature of the enzyme were 7.4～7. 8 and 60℃, respectively. The Km was 19.3 mmol / L. The enzyme required Mg2+ as a cofactor for hydrolysis of pyrophosphate and was inhibited by Ca2+, Hg2+, Pb2+, Mn2+.     

ADP, chloride ion, and metal ion binding to bovine brain glutamine synthetase

The binding of divalent cations and nucleotide to bovine brain glutamine synthetase and their effects on the activity of the enzyme were investigated. In ADP-supported gamma-glutamyl transfer at pH 7.2, kinetic analyses of saturation functions gave [S]0.5 values of approximately 1 microM for Mn2+, approximately 2 mM for Mg2+, 19 nM for ADP.Mn, and 7.2 microM for ADP.Mg. The method of continuous variation applied to the Mn2+-supported reaction indicated that all subunits of the purified enzyme express activity when 1.0 equiv of ADP is bound per subunit. Measurements of equilibrium binding of Mn2+ to the enzyme in the absence and presence of ADP were consistent with each subunit binding free Mn2+ (KA approximately equal to 1.5 X 10(5) M-1) before binding the Mn.ADP complex (KA' approximately equal to 1.1 X 10(6) M-1). The binding of the first Mn2+ or Mg2+ to each subunit produces structural perturbations in the octameric enzyme, as evidenced by UV spectral and tryptophanyl residue fluorescence changes. The enzyme, therefore, has one structural site per subunit for Mn2+ or Mg2+ and a second site per subunit for the metal ion-nucleotide complex, both of which must be filled for activity expression. Chloride binding (KA' approximately equal to 10(4) M-1) to the enzyme was found to have a specific effect on the protein conformation, producing a substantial (30%) quench of tryptophanyl fluorescence and increasing the affinity of the enzyme 2-4-fold for Mg2+ or Mn2+. Arsenate, which activates the gamma-glutamyl transfer activity by binding to an allosteric site, and L-glutamate also cause conformational changes similar to those produced by Cl- binding. Anion binding to allosteric sites and divalent metal ion binding at active sites both produce tryptophanyl residue exposure and tyrosyl residue burial without changing the quaternary enzyme structure.     

Transient state in the phosphorylation of sodium- and potassium- transport adenosine triphosphatase by adenosine triphosphate

The rate of phosphorylation of sodium and potassium ion-transport adenosine triphosphatase by 10 microM [gamma-32P]ATP was much slower with Ca2+ than with Mg2+ (0.13-10 mM) in the presence of 16 to 960 mM Na+ at 0 degrees C and pH 7.4. In the presence of a fixed concentration of Mg2+ or Ca2+, the rate became slower with increasing Na+ concentration. When the Na+ concentration was fixed, the rate became slower with decreasing divalent cation concentration. Sodium ions appear to antagonize the divalent cation in the phosphorylation to slow its rate. In the presence of 1 mM Ca2+ and 126 or 270 mM Na+, the rate was slow enough to permit the manual addition of a chasing solution at various times before the phosphorylation reached the steady state. Therefore, we studied the time-dependent change of the sensitivity to ADP or to K+ of the phosphoenzyme by a chase with unlabeled ATP containing ADP or K+ during the time range from the transient to the steady state of the phosphorylation. The ADP sensitivity decreased and the K+ sensitivity increased with the progress of the phosphorylation. With 270 mM Na+, the phosphoenzyme found at 1 s, when its amount was 5.5% of the maximum level, was virtually completely sensitive to ADP. Under these conditions, it was concluded that the form of the phosphoenzyme initially produced from the enzyme.ATP complex has ADP sensitivity and that the phosphoenzyme acquires K+ sensitivity later. The initially produced ADP-sensitive phosphoenzyme partially lost its normal instability and sensitivity upon adding a chelating agent, probably because of dissociation of a divalent cation from the phosphoenzyme.