Binding of divalent cation to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase.

In order to study the action of the divalent cation which is essential for phosphorylation of sodium- and potassium-transport adenosine triphosphatase, magnesium ion, the normal ligand, was replaced with calcium ion, which had properties diffeerent from those of Mg2+, Mn2+, Fe2+, Co2+, Ni2+, or Zn2+. Phosphorylation of the enzyme from ATP at pH 7.4 in the presence of Na+ and Ca2+ yielded a Ca.phosphoenzyme (60% of the maximal level) with a normal rate of dephosphorylation following a chase with unlabeled Ca.ATP (PK = 0.092S-1 at 0 degrees C). In contrast, after a chase by a chelator, namely ethylenediaminetetraacetic acid, 1,2-cyclohexylenedinitrilotetraacetic acid, or ethylene glycol bis-(beta-aminoethyl ether)N,N'-tetraacetic acid, dephosphorylation slowed within 5 s and half of the initial phosphoenzyme remained with a stability about 5-fold greater than normal. Three states of the phosphoenzyme were distinguished according to their relative sensitivity to ADP or to K+ added during a chase. Normally prepared Mg.phosphoenzyme was sensitive to K+ but not to ADP; Ca.phosphoenzyme was sensitive either to ADP or to K+; and the stabilized phosphoenzyme prepared from Ca.phosphoenzyme by addition of a chelator was sensitive neither to ADP nor to K+ nor to both together. Addition of Ca2+ to the stabilized phosphoenzyme restored the reactivity to that of Ca.phosphoenzyme. Addition of Mg2+ to the stabilized phosphoenzyme changed the reactivity to that of Mg.phosphoenzyme. Therefore, this unreactive, stabilized state of the phosphoenzyme appeared to be a divalent cation-free phosphoenzyme. With respect to sensitivity to ouabain, Ca.phosphoenzyme was as sensitive as Mg.phosphoenzyme but calcium-free phosphoenzyme was much less sensitive. It was concluded that the divalent cation required for phosphorylation normally remains tightly bound to the phosphoenzyme and is required for normal reactivity. Calcium ion was almost unique in dissociating relatively easily from the phosphoenzyme. Strontium ion appeared to act similarly to Ca2+.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Rapid release of 42K and 86Rb from an occluded state of the Na,K-pump in the presence of ATP or ADP

We have measured the time course of release of 42K and 86Rb from an occluded state of the Na,K-pump using a rapid filtration apparatus. We have found that at 20 degrees C and in the presence of ATP, 42K is released with a rate constant of approximately 45 s-1 and 86Rb with a rate constant of approximately 20 s-1; both ATP and ADP are effective at a low affinity site (Kd approximately 0.3 and 1 mM, respectively) with the rate of deocclusion being only half as great in ADP as in ATP. Mg2+ stimulates 2-fold at low concentrations probably by forming MgATP, and free Mg2+ is strongly inhibitory at high concentrations (Kd approximately 10 mM). Mg2+ also decreases the affinity for ATP, and the data are consistent with mixed type inhibition; from the analysis the dissociation constant is approximately 1 mM for the inhibitory Mg2+ and the Rb+-occluded form without ATP. The rate of 42K or 86Rb release increases monotonically with pH while ATPase activity decreases above pH 8, so that deocclusion is not rate-limiting in the overall cycle at high pH. This is reflected by a convergence of the rate of Na,K-ATPase and Na,Rb-ATPase activities at high pH and by a decrease in the observed steady-state level of the occluded 86Rb intermediate at high pH. K+, Rb+, Na+, and Cs+, but not Li+, increase the rate of 42K and 86Rb release at constant ionic strength, presumably at sites other than the transport sites. The spontaneous rate of deocclusion is only approximately 0.1 s-1 at low ionic strength in the absence of nucleotides, and it is increased markedly by all cations tested except Li+. Overall the data are consistent with deocclusion as a rate-limiting step in the Na,K-pump cycle.     

Purification and characterizaton of plastidic pyruvate kinase from developing seeds of Brassica campestris L.

Plastidic pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was purified to near homogeneity as judged by native PAGE with about 4% recovery from developing seeds of Brassica campestris using (NH4)2SO4 fractionation, DEAE-cellulose chromatography, gel filtration through Sepharose-CL-6B and affinity chromatography through reactive blue Sepharose-CL-6B. The purified enzyme having molecular mass of about 266 kDa was quite stable and showed a broad pH optimum between pH 6.8-7.8. Typical Michaelis-Menten kinetics was obtained for both the substrates with K(m) values of 0.13 and 0.14 mM for PEP and ADP, respectively. The enzyme could also utilize CDP, GDP or UDP as alternative nucleotide to ADP, but with lower Vmax and higher K(m). The enzyme had an absolute requirement for a divalent and a monovalent cation for activity and was inhibited by oxalate, fumarate, citrate, isocitrate and ATP, and activated by AMP, aspartate, 3-PGA, tryptophan and inorganic phosphate. ATP inhibited the enzyme competitively with respect to PEP and non-competitively with respect to ADP. Similarly, oxalate inhibition was also of competitive type with respect to PEP and non-competitive with respect to ADP. This inhibition by either ATP or oxalate was not due to chelation of Mg2+, as the inhibition was not relieved on increasing Mg2+ concentration even upto 30 mM. Initial velocity and product inhibition studies demonstrated the reaction mechanism to be compulsory ordered type. The enzyme seems to be regulated synergistically by ATP and citrate.     

Critical role of specific ions for ligand-induced changes regulating pyruvate dehydrogenase kinase isoform 2

In the complete absence of K+ and phosphate (Pi), pyruvate dehydrogenase kinase isoform 2 (PDHK2) was catalytically very active but with an elevated Km for ATP, and this activity is insensitive to effector regulation. We find that K+ or 5-fold lower levels of NH4+ markedly enhanced quenching of Trp383 fluorescence of PDHK2 by ADP and ATP. K+ binding caused an approximately 40-fold decrease in the equilibrium dissociation constants (Kd) for ATP from approximately 120 to 3.0 microM and an approximately 25-fold decrease in Kd for ADP from approximately 950 to 38 microM. Linked reductions in Kd of PDHK2 for K+ were from approximately 30 to approximately 0.75 mM with ATP bound and from approximately 40 to approximately 1.7 mM with ADP bound. Without K+, there was little effect of ADP on pyruvate binding, but with 100 mM K+ and 100 microM ADP, the L0.5 of PDHK2 for pyruvate was reduced by approximately 14 fold. In the absence of K+, Pi had small effects on ligand binding. With 100 mM K+, 20 mM Pi modestly enhanced binding of ADP and hindered pyruvate binding but markedly enhanced the binding of pyruvate with ADP; the L0.5 for pyruvate was specifically decreased approximately 125-fold with 100 microM ADP. Pi effects were minimal when NH4+ replaced K+. We have quantified coupled binding of K+ with ATP and ADP and elucidated how linked K+ and Pi binding are required for the potent inhibition of PDHK2 by ADP and pyruvate.     

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.     

Kinetic mechanism of ATP synthesis catalyzed by mitochondrial Fo x F1-ATPase

Initial rates of succinate-dependent ATP synthesis catalyzed by submitochondrial particles from bovine heart substoichiometrically coupled with oligomycin were found to have hyperbolic dependencies on contents of Mg x ADP, free Mg2+, and phosphate. The results suggest that Mg x ADP complex and free phosphate are true substrates of the enzyme; and an unordered ternary complex of Fo x F1-ATPase, Mg x ADP, and phosphate is generated during the catalysis. The presence of free Mg2+ is required for the reaction. Mg2+ was a noncompetitive activator of ATP synthesis relative to Mg x ADP and a competitive activator relative to phosphate. The decrease in steady-state values of Deltamu(H)+ (by the inhibition of succinate oxidase with malonate) results in the decreased value of Vmax and in a slight decrease in Km for the substrates and Mg2+ without changes in affinity for the substrates. Based on these results, a kinetic scheme of ATP synthesis is proposed.     

Inhibition Effect of Zn2+ on Cytosolic Pyruvate Kinase in Passiflora edulis Leaves

Cytosolic pyruvate kinase (PKc) activity in both mature and young leaves of Passiflora eclulis Smis was inhibited by Zn2+, and the severity of inhibition was dependant upon the concentration of Mg2+ present in the solution . Some differences in the inhibitory kinetics existed between PKc in mature and young leaves. Zn2+ showed anti-competitive inhibition of PKc in both mature and young leaves with respect to PEP and ADP, Ki′ (PEP) were 0. 059 mmol/L and 0. 038 mmol/L, and Ki′ (ADP) were 0. 074 mmol/L and 0.02 mmol/L respectively for PKc in mature and young leaves. Ki (Mg2+) of Zn2+ on PKc in both mature and young leaves were 0. 016 mmol/L and 0. 008 mmol/L respectively. These results further substantiated our previous conclusion that the in regulatory properties of PKc in mature leaves were different from those in young leaves.     

Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli

The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate-binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg(2+)) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20 degrees C, 5 mM pyruvate (with 2 mM Mg(2+)) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg(2+) is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 10(8) to < 5 x 10(5) or 3 x 10(7) M(-1), respectively. Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg(2+) also increases domain coupling and the dimerization constant approximately 42-fold. With 2 mM Mg(2+) at 15-25 degrees C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K(A)' approximately 10(6) M(-1) (deltaG' = -8.05 +/- 0.05 kcal/mole and deltaH = +3.9 kcal/mole at 20 degrees C); deltaC(p) = -0.33 kcal K(-1) mole(-1). The binding of PEP to EI(H189A) is synergistic with that of Mg(2+). Thus, physiological concentrations of PEP and Mg(2+) increase, whereas pyruvate and Mg(2+) decrease the amount of dimeric, active, dephospho-enzyme I.     

Specific ion influences on self-association of pyruvate dehydrogenase kinase isoform 2 (PDHK2), binding of PDHK2 to the L2 lipoyl domain, and effects of the lipoyl group-binding site inhibitor, Nov3r

Association of the PDHK2 and GST-L2 (glutathione-S-transferase fused to the inner lipoyl domain (L2) of dihydrolipoyl acetyltransferase (E2)) dimers was enhanced by K+ with higher affinity K+ binding than occurs at the PDHK2 active site. Supporting a distinct K+ binding site, the NH4+ ion did not effectively replace K+ in aiding GST-L2 binding. With 50 mM K+, Pi enhanced interference by ADP, ATP, or pyruvate of PDHK2 binding to GST-L2. The inclusion of Pi with ADP or ATP plus pyruvate greatly hindered PDHK2 binding to GST-L2 and promoted PDHK2 forming a tetramer. Reciprocally, GST-L2 interference with ATP/ADP binding also required elevated K+ and was increased by Pi. Potent inhibition by Nov3r of E2-activated PDHK2 activity (IC50 of approximately 7.8 nM) required elevated K+ and Pi. Nov3r only modestly inhibited the low activity of PDHK2 without E2. By binding at the lipoyl group binding site, Nov3r prevented PDHK2 binding to E2 and GST-L2. Nov3r interfered with high-affinity binding of ADP and pyruvate via a Pi-dependent mechanism. Thus, GST-L2 binding to PDHK2 is supported by K+ binding at a site distinct from the active site. Pi makes major contributions to ligands interfering with PDHK2 binding to GST-L2, the conversion of PDHK2 dimer to a tetramer, and Nov3r (an acetyl-lipoate analog) interfering with binding of ADP and pyruvate. Pi is suggested to facilitate transmission within PDHK2 of the stimulatory signal of acetylation from the distal lipoyl-group binding site to the active site.     

Steady state kinetics of pyruvate kinase from muscle]

The dependence of the forward pyruvate kinase reaction on the concentrations of ADP and Mg2+ was studied. It was shown that high total ADP concentrations (2-15 mM) exceeding total Mg2+ concentration in the medium lead to the inhibition of the pyruvate kinase reaction, whereas relatively high Mg2+ concentrations (up to 15 mM) do not cause any inhibition. The kinetics of the reaction can be described in the best way by a scheme incorporating the active PEP . E . Mg2+ . Mg . ADP complex and dead-end complexes containing free ADP. An analysis of the experimental data allows to determine all coefficients of the rate equation and to calculate the values of all kinetic parameters. The values of the constants obtained were used for mathematical simulation of the reaction on the basis of the kinetic scheme given. The mathematical model obtained describes satisfactorily the experimentally determined dependences, which is indicative of the correctness of the model.     

Reaction mechanism of calcium-ATPase of sarcoplasmic reticulum. Substrates for phosphorylation reaction and back reaction, and further resolution of phosphorylated intermediates

Reaction of the purified Ca2+-ATPase of sarcoplasmic reticulum at 0 degrees C at low [gamma-32P]ATP (0.1 to 0.67 microM) and enzyme (0.025 to 0.24 microM) concentration in the presence of 0.11 to 30 mM Ca2+ without added Mg2+ has resulted in the formation of phosphorylated intermediate (EP:maximal level of EP = 0.45 mol/mol of enzyme) at a very slow rate. Under these conditions, the reaction steps in which EP decomposition takes place are completely prevented. This has permitted us to study the EP formation reaction and its reversal specifically, with a considerably improved time resolution. An apparent rate constant of EP formation (Vf) increases in parallel with the concentration of Ca . ATP, but not with those of Mg . ATP, or of protonated or fully ionized free ATP. This suggests that Ca . ATP is the substrate under these conditions. If Co2+ or Mn2+ are in excess over the other ions during the reaction, Vf varies in parallel with [Co . ATP] or [Mn . ATP]. Thus, it appears that either Ca2+, Co2+, or Mn2+ can be complexed with ATP to form the effective substrate. An apparent rate constant of the back reaction of EP initiated by addition of ADP to EP (Vr) increases in proportion to [ADP] or [H . ADP], but is inhibited by increasing concentrations of the ADP complex with Ca2+ or Mg2+, indicating that free ADP or protonated ADP, or both, are actual substrates for the back reaction of EP. These results suggest a new type of site to which the metal moiety of metal . ATP complex remains bound after the release of ADP from the enzyme. An acid-stable phosphorylated intermediate (EP) produced in the presence of high Ca2+ concentrations (e.g. 0.11 mM) without added Mg2+ does not decompose spontaneously, and the major portion (approximately 90%) of this EP (EPD+) reacts with ADP to form ATP (ADP-sensitive). Upon chelating Ca2+ with ethylene glycol bis(beta-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA), EPD+ is converted to another form of EP (EPD-), which is unreactive with ADP (or ADP-insensitive). Addition of Mg2+, after initiation of the reaction leading to EPD- by EGTA, results in rapid production of Pi from a portion of EPD- with KMg approximately equal to 3.3 x 10(3) M-1. The fraction of EPD- that is Mg2+-sensitive (EPD-,M+) increases with reaction time at a much slower rate than the Mg2+-insensitive portion of EPD- (EPD-,M-). These results suggest that the enzyme reaction involves the sequential formation of at least three forms of acid-stable EP, viz. in the order of formation, EPD+, EPD-,M-, and EPD-,M+. The equilibrium between EPD+ and EPD-,M- is shifted by higher [K+] and [Ca2+] towards EPD+.     

Pyruvate kinase in the bovine adrenal cortex

Pyruvate kinase from bovine adrenal cortex was purified to an electrophoretically homogeneous state. The molecular weight of the native enzyme is about 230 000, that of one subunit is 57 000. The maximal values of the pyruvate kinase initial reaction rate were obtained in 50 mM imidazole-acetate buffer within the pH range of 6.8 to 7.0. The curve of the initial pyruvate kinase reaction rate versus phosphoenolpyruvate (PEP) and ADP concentrations is hyperbolic and obeys the Michaelis-Menten kinetics with Km for PEP and ADP of 0.055 X 10(-3) M and 0.25 X 10(-3) M, respectively. The enzyme is activated by Mn2+ and Co2+ by 43 and 38%, respectively. IDP, GDP, and UDP may be used as analogs of ADP. The enzyme is not activated by fructose-1.6-diphosphate and is inhibited by L-phenylalanine and ATP.     

Rat-brain pyruvate kinase: Purification and effects of lithium

Purified pyruvate kinase was prepared from pooled brains obtained from untreated rats. Its properties suggest that it is similar to type 'M' pyruvate kinase. Lithium inhibition was demonstrated at pharmacologically significant lithium concentrations (7%-12% at 2 mmol 1-1 Li) and this was similar in character to that previously seen in rabbit muscle pyruvate kinase, namely noncompetitive with respect to phosphoenol pyruvate, K+, and Mg2+ but competitive with ADP.     

Use of methanethiolation to investigate the catalytic role of sulphydryl groups in rabbit skeletal muscle pyruvate kinase.

Incubation of rabbit skeletal muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) with methyl methanethiosulphonate resulted in the time- and inhibitor concentration-dependent loss of enzyme activity. Substrates or products of the catalytic reaction prevented the loss of activity caused by methanethiolation. Their effectiveness as protecting agents was placed in the order ADP greater than ATP greater than Mg2+ greater than phosphoenolpyruvate greater than pyruvate. The essential catalytic cation, K+, had no effect on the methanethiolation reaction. [Me-3H]Methanethiosulphonate modified all the available cysteine thiol groups which correlated to the incorporation of four SC3H3 groups per protomer. Four radioactive peptides were obtained on tryptic peptide mapping. When methanethiolation was carried out in the presence of Mg2+ alone or with Mg2+ and ATP together, then only three SC3H3 groups were incorporated into each subunit. If MgATP protected methanethiolated pyruvate kinase was reacted with iodo[2-3H]acetic acid then 1.37 +/- 0.2 groups per protomer were carboxymethylated. 70% of the radioactivity was located in a single peptide on tryptic peptide mapping. This peptide was isolated and contained the segment carboxymethyl cysteine (Glx, Asx, Ser) Arg. Collectively these data indicate that although all thiol groups are equally accessible to methyl methanethiosulphonate, only a single thiol group participates in the catalytic event. An additional role in the maintenance of structure for this thiol group was also shown in studied of reduction and thermal denaturation of the enzyme.     

Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.     

ATP/ADP exchange activity of gastric (H+ +K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the (H+ +K+)-ATPase by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to P1, P5-di(adenosine-5') pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration (K0.5=116 microM) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+.     

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes.

The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3)J(H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+- and Mn2+- activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t(1/2) = 0.7 min).     

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.