Conformations and arrangement of substrates at active sites of ATP-utilizing enzymes

The phosphoryl transferring enzymes pyruvate kinase, cAMP-dependent protein kinase and the pyrophosphoryl transferring enzyme PP-Rib-P synthetase utilize the beta, gamma bidentate metal--ATP chelate (delta-isomer) as substrate, as determined with substitution-insert CrIIIATP or CoIII(NH3)4ATP complexes. In addition, these enzymes bind a second divalent cation, which is an essential activator for pyruvate kinase and PP-Rib-P synthetase and an inhibitor of protein kinase. The enzyme-bound metal has been used as a paramagnetic reference point in T1 measurements to determine distances to the protons and phosphorus atoms of the bound nucleotide and acceptor substrates. These distances have been used to construct models of the conformations of the bound substrates. The activating metal forms a second sphere complex of the metal-nucleotide substrate on pyruvate kinase and PP-Rib-P synthetase while the inhibitory metal directly coordinates the polyphosphate chain of the metal-nucleotide substrate on protein kinase. Essentially no change is found in the dihedral angle at the glycosidic bond of ATP upon binding to pyruvate kinase (chi = 30 degrees), an enzyme of low base specificity, but significant changes in the torsional angle of ATP occur on binding to protein kinase (chi = 84 degrees) and PP-Rib-P synthetase (chi = 62 degrees), enzymes with high adenine-base specificity. Intersubstrate distances, measured with tridentate CrATP or beta, gamma bidentate CrAMPPCP as paramagnetic reference points, have been used to deduce the distance along the reaction coordinate on each enzyme. The reaction coordinate distances on pyruvate kinase (# +/- 1 A) and PP-Rib-P synthetase (not less than 3.8 A) are consistent with associative mechanisms, while that on protein kinase (5 +/- 0.7 A) allows room for a dissociative mechanism.     

Nuclear Overhauser effect studies of the conformations of MgATP bound to the active and secondary sites of muscle pyruvate kinase

MgATP binds both at the active site (site 1) and at a secondary site (site 2) on each monomer of muscle pyruvate kinase as previously found by binding studies and by X-ray analysis. Interproton distances on MgATP bound at each site have been measured by the time-dependent nuclear Overhauser effect in the absence and presence of phosphoenolpyruvate (P-enolpyruvate), which blocks ATP binding at site 1. Interproton distances at site 2 are consistent with a single conformation of bound ATP with a high antiglycosidic torsional angle (chi = 68 +/- 10 degrees) and a C3'-endo ribose pucker (delta = 90 +/- 10 degrees). Interproton distances at site 1, determined in the absence of P-enolpyruvate by assuming the averaging of distances at both sites, cannot be fit by a single adenine-ribose conformation but require the contribution of at least three low-energy structures: 62 +/- 10% low anti (chi = 30 degrees), C3'-endo; 20 +/- 8% high anti (chi = 55 degrees), O1'-endo; and 18 +/- 8% syn (chi = 217 degrees), C2'-endo. Although a different set of ATP conformations might also have fit the interproton distances, the mixture of conformations used also fits previously determined distances from Mn2+ to the protons of ATP bound at site 1 [Sloan, D. L., & Mildvan, A. S. (1976) J. Biol. Chem. 251, 2412] and is similar to the adenine-ribose portion of free Co(NH3)4ATP, which consists of 35% low anti, 51% high anti, and 14% syn [Rosevear, P. R., Bramson, H. N., O'Brian, C., Kaiser, E. T., & Mildvan, A. S. (1983) Biochemistry 22, 3439].(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of the alpha, beta, gamma-tridentate manganese complex of adenosine 5'-triphosphate cocrystallized with 2,2'-dipyridylamine

The 1:1:1 complex of Mn2+, ATP, and 2,2'-dipyridylamine (DPA) crystallizes as Mn-(HATP)2.Mn(H2O)6.(HDPA)2.12H2O in the orthorhombic space group C222(1) with unit cell dimensions a = 10.234 (3) A, b = 22.699 (3) A, and c = 31.351 (4) A. The structure was solved by the multisolution technique and refined by the least-squares method to a final R index of 0.072 using 3516 intensities. The structure is composed of two ATP molecules sharing a common manganese atom. The metal exhibits alpha, beta, gamma coordination to the triphosphate chains of two dyad-related ATP molecules, resulting in a hexacoordinated Mn2+ ion surrounded by six phosphate groups. The metal to oxygen distances are 2.205 (6), 2.156 (4), and 2.144 (5) A for the alpha-, beta-, and gamma-phosphate groups, respectively. No metal-base interactions are observed. There is a second hexaaqua-coordinated Mn2+ ion that is also located on a dyad axis. The hydrated manganese ions sandwich the phosphate-coordinated manganese ions in the crystal with a metal-metal distance of 5.322 A. The ATP molecule is protonated on the N(1) site of the adenine base and exhibits the anti conformation (chi = 66.0 degrees). The ribofuranose ring is in the 2/3 T conformation with pseudorotation parameters P = 179 (1) degrees and tau m = 34.1 (6) degrees. The adenine bases form hydrogen-bonded self-pairs across a crystallographic dyad axis and stack with both DPA molecules to form a column along the dyad. The structure of the metal-ATP complex provides information about the possible metal coordination, conformation, and environment of the nucleoside triphosphate substrate in the enzyme.     

Nucleotide binding to tubulin-investigations by nuclear magnetic resonance spectroscopy

In an attempt to distinguish between the interaction of GTP and ATP with tubulin dimer, high-resolution ¹H- and ³¹P-NMR experiments have been carried out on the nucleotides in the presence of tubulin. The location of the ATP binding sites on the protein in relation to the GTP sites is still not clear. Using NMR spectroscopy, we have tried to address this question. Evidence for the existence of a site labelled as X-site and another site (labelled as L-site for both the nucleotides on tubulin has been obtained. It is suggested that this X-site is possibly the putative E-site. In order to gain further insight into the nature of these sites, the Mg(II at the N-site has been replaced by Mn(II and the paramagnetic effect of Mn(II on the linewidth of the proton resonances of tubulin-bound ATP and GTP has been studied. The results show that the L-site nucleotide is closer to the N-site metal ion compared to the X-site nucleotide. On the basis of these results, it is suggested that the L-site of ATP is distinct from the L-site of GTP while the X-site of both the nucleotides seems to be same. By using the paramagnetic effect of the metal ion, Mn(II), at the N-site on the relaxation rates of tubulin-bound ATP at L-site, distances of the protons of the base, sugar and phosphorous nuclei of the phosphorous moiety of ATP, from the N-site metal ion have been mapped. The base protons are 2 0.7–1 nm distant from the N-site metal ion, while the protons of the sugar are 2 0.8-1 nm from this metal ion site. On the other hand, the phosphorous nuclei of the phosphate groups are somewhat nearer (2 0.4–0.5 nm from the N-site metal ion.     

Dual divalent cation requirement for activation of pyruvate kinase; essential roles of both enzyme- and nucleotide-bound metal ions.

Rabbit muscle pyruvate kinase requires two divalent cations per active site for catalysis of the enolization of pyruvate in the presence of adenosine 5'-triphosphate (ATP). One divalent cation is bound directly to the enzyme and forms a second sphere complex with the bound ATP (site 1). The second divalent cation is directly coordinated to the phosphoryl groups of ATP and does not interact with the enzyme (site 2). The essential role of the divalent cation at site 1 is shown by the requirement for Mg2+ or Mn2+ for the enolization of pyruvate in the presence of the substitution inert Cr3+-ATP complex. The rate of detritiation of pyruvate shows a hyperbolic dependence of Mn2+ concentration in the presence of high concentrations of enzyme and Cr3+-ATP. A dissociation constant for Mn2+ from the pyruvate kinase-Mn2+-ATP-Cr3+-pyruvate complex of 1.3 +/- 0.5 muM is determined by the kinetics of detritiation of pyruvate and by parallel Mn2+ binding studies using electron paramagnetic resonance. The essential role of the divalent cation at site 2 is shown by the sigmoidal dependence of the rate of detritiation of pyruvate on Mn2+ concentration in the presence of high concentrations of enzyme and ATP yielding a dissociation constant of 29 +/- 9 muM for Mn2+ from site 2. This value is similar to the dissociation constant of the binary Mn-ATP complex (14 +/- 6 muM) determined under similar conditions. The rate of detritiation of pyruvate is proportional to the concentration of the pyruvate kinase-Mn2+-ATP-Mn2+-pyruvate complex, as determined by parellel kinetic and binding studies. Variation of the nature of the divalent cation at site 1 in the presence of CrATP causes only a twofold change in the rate of detritiation of pyruvate which does not correlate with the pKa of the metal-bound water. Variation of the nature of the divalent cation at both sites in the presence of ATP causes a sevenfold variation in the rate of detritiation or pyruvate that correlates with the pKa of the metal-bound water. The greater rate of enolization observed with CrATP fits this correlation, indicating that the electrophilicity of the nucleotide bound metal (at site 2) determines the rate of enolization of pyruvate.     

Orotate phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase from yeast: nuclear magnetic relaxation studies of the structures of enzyme-bound phosphoribosyl 1-pyrophosphate

Nuclear magnetic relaxation rate measurements have been performed on the protons and phosphorus atoms of phosphoribosyl 1-pyrophosphate (PRibPP) in the presence and absence of paramagnetic chromium(III), cobalt(II), and manganese(II) ions. The longitudinal relaxation rates were then used to calculate interatomic distances between the magnetic nuclei and these paramagnetic probes, from which was devised a conformation of the PRibPP-metal ion complex in solution. Thereafter, the experiments were accomplished in the presence of Mn(II) and a series of orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) concentrations, and from these data were estimated the distances between Mn(II) and the PRibPP nuclei at the active sites of these two enzymes from yeast. Comparisons between the Mn(II)-PRibPP conformation in solution and this structure at the active sites of OPRTase and HGPRTase revealed that the metal ion remained coordinated with the pyrophosphate group of PRibPP in all instances, whereas the overall distances between the ribose ring and Mn(II) at the enzyme active sites were approximately 1 A further from the metal ion. Model building studies also revealed that the 5'-phosphate group of PRibPP is positioned directly over the ribose ring in solution and at the OPRTase and HGPRTase active sites and may protect the 1'-carbon of PRibPP against on-line displacements of pyrophosphate under these conditions, where the PRibPP-to-Mn(II) concentration ratio is greater than 2000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromium(III)-adenosine triphosphate as a paramagnetic probe to determine intersubstrate distances on pyruvate kinase. Detection of an active enzyme-metal-ATP-metal complex.

The interaction of CrATP, a stable, substitution-inert, paramagnetic tridentate complex of ATP, with muscle pyruvate kinase has been studied by measuring the effects of CrATP on the kinetics of pyruvate enolization and on the longitudinal nuclear magnetic relaxation rate (1/T1) of the protons of water and the protons and carbon atoms of pyruvate to investigate the existence and activity of bimetallic enzyme-M(II)-CrATP complexes and to determine intersubstrate distances on a kinase. The paramagnetic effect of CrATP on 1/T1 of water protons is enhanced upon complexation with the enzyme. Titrations of the enzyme with CrATP yielded characteristic enhancements of 1/T1 for the binary enzyme-CrATP, ternary enzyme-Mg(II)-crATP, and quaternary enzyme-Mg(II)-crATP-pyruvate complexes of 3.5, 1.7, and 1.2 and dissociation constants of CrATP of 400, 200, and 200 muM, respectively. From the frequency dependence of 1/T1, the number of fast exchanging water protons in the coordination spheres of Cr(III) is approximately 6 in CrATP and in both the ternary enzyme-Mg(II)-CrATP complex and the quaternary enzyme-Mg(II)-CrATP-pyruvate complex. The paramagnetic effect of enzyme-bound Mn(II) on 1/T1 of water protons decreases upon the addition of CrATP. Titration of the binary enzyme-Mn(II) complex with CrATP decreases the characteristic enhancement due to Mn(II) from 24 +/- 3 to 6 +/- 1. Titration of the ternary eznyme-Mn(II)-pyruvate complex with CrATP decreases the enhancement from 6 +/- 1 to 0.5 +/- 0.1. The affinity of the enzyme for Mn(II) is increased 2-fold upon binding of CrATP as indicated by decreases in the amplitude of the EPR spectrum of free Mn(II). The dissociation constants of CrATP from the enzyme-Mn(II)-CrATP complex, the enzyme-CrATP-pyruvate complex, and the enzyme-Mn(II)-CrATP-pyruvate complex are all 200 muM. The observed titration behavior, the characteristic enhancement values, the tightening by Mg(II) of the binding of CrATP to the enzyme, and the tightening of the binding of Mn(II) to the enzyme by CrATP establish the existence of enzyme-M(II)-CrATP and enzyme-M(II)-CrATP-pyruvate complexes containing two cations, Mg(II) or Mn(II) and Cr(III), at the active site.     

NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme

Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual consistency of interproton and Cr3+ to proton distances obtained in metal-ATP complexes of both the enzyme and the peptide suggests that the conformation of the peptide is very similar to that of residues 1-45 of the enzyme. When this was assumed to be the case and when molecular models and a computer graphics system were used, MgATP could be fit into the X-ray structure of adenylate kinase in a unique manner such that all of the distances determined by NMR were accommodated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural and exchange properties of "Co(III)-phenanthroline-ATP": a labeling reagent for the active site of ATPases.

This paper reports on 1H and 31P NMR as well as EPR measurements of the labeling reagent of ATPase sites, "Co(III)-(phen)-ATP." This complex is found to be paramagnetic, as deduced both from its EPR spectrum and from the significant broadening, though almost unshifted, proton and phosphorus resonances. This paramagnetism is a result of the incorporation of the superoxide free-radical anion in the coordination sphere of the trivalent cobalt ion. Evidence for the presence of superoxide in the complex is based on competition experiments with cyanide, which is able to displace the superoxide anion. The latter was identified by its inducing effect on the photoreactivity of luminol. The displacement of superoxide by cyanide was accompanied by the abolition of the paramagnetism of the complex. The relative distances between the protons and phosphorus atoms of ATP and the superoxide anion in the complex were calculated using the NMR line-broadening data. Structural models compatible with the experimental results are proposed. Under conditions of excess of adenine nucleotides or phenanthroline, the coordinated ATP molecule becomes exchangeable. This phenomenon is attributed to the labilization of the cobaltic ion ligands induced by the superoxide anion.     

Quantitative determination of the conformation of ATP in aqueous solution using the lanthanide cations as nuclear-magnetic-resonance probes.

Chemical shift perturbations of the eight 1H resonances and of the three 31P resonances in the nuclear magnetic resonance spectra of ATP in 2H2O, pH 6.0, have been induced by specifically bound lanthanide cations Ln3+ (Ln = Pr, Nd, Eu, Yb). After separation of contact (through bond) perturbations the resultant through-space shifts, which are found to have axial symmetry, are used in an analysis of the conformation of the Ln3+ -ATP complex. A computer program was used to search for the conformations of the molecule which fit the nuclear magnetic resonance data. The "best" solutions obtained represent a small closely interrelated family of conformations. Effects of the cation Gd3+ on the longitudinal relaxation rates of five of the protons of ATP were also measured and used to confirm the conformational family. One of these conformations corresponds closely to one of the crystal structure forms, with an anti arrangement of the base-ribose unit and and a right-hand helical phosphate chain folded towards the adenine part of the molecule. The lanthanide ion binds predominantly to the beta and gamma phosphates and does not interact with the purine ring, these two centres being separated by at least one water molecule.     

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.     

31P and 1H NMR studies of the structure of enzyme-bound substrate complexes of lobster muscle arginine kinase: relaxation measurements with Mn(II) and Co(II)

The paramagnetic effects of Mn(II) and Co(II) on the spin-lattice relaxation rates of 31P nuclei of ATP and ADP and of Mn(II) on the spin-lattice relaxation rate of the delta protons of arginine bound to arginine kinase from lobster tail muscle have been measured. Temperature variation of 31P relaxation rates in E.MnADP and E.MnATP yields activation energies (delta E) in the range 6-10 kcal/mol. Thus, the 31P relaxation rates in these complexes are exchange limited and cannot provide structural information. However, the relaxation rates in E.CoADP and E.CoATP exhibit frequency dependence and delta E values in the range 1-2 kcal/mol; i.e., these rates depend upon 31P-Co(II) distances. These distances were calculated to be in the range 3.2-4.5 A, appropriate for direct coordination between Co(II) and the phosphoryl groups. The paramagnetic effect of Mn(II) on the 1H spin-lattice relaxation rate of the delta protons of arginine in the E.MnADP.Arg complex was also measured at three frequencies (viz., 200, 300, and 470 MHz). These 1H experiments were performed in the presence of sufficient excess of arginine to be observable over the protein background but with MnADP exclusively in the enzyme-bound form so that the enhancement in the relaxation rates of the delta protons of arginine arises entirely from the enzyme-bound complex. Both the observed frequency dependence of these rates and the delta E less than or equal to 1.0 +/- 0.3 kcal/mol indicate that this rate depends on the 1H-Mn(II) distances.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells

Extracellular ATP synthesis on human umbilical vein endothelial cells (HUVECs) was examined, and it was found that HUVECs possess high ATP synthesis activity on the cell surface. Extracellular ATP generation was detected within 5 s after addition of ADP and inorganic phosphate and reached a maximal level at 15 s. This type of ATP synthesis was almost completely inhibited by mitochondrial H(+)-ATP synthase inhibitors (e.g., efrapeptins, resveratrol, and piceatannol), which target the F(1) catalytic domain. Oligomycin and carbonyl cyanide m-chlorophenylhydrazone, but not potassium cyanide, also inhibited extracellular ATP synthesis on HUVECs, suggesting that cell surface ATP synthase employs the transmembrane electrochemical potential difference of protons to synthesize ATP as well as mitochondrial H(+)-ATP synthase. The F(1)-targeting H(+)-ATP synthase inhibitors markedly inhibited the proliferation of HUVECs, but intracellular ATP levels in HUVECs treated with these inhibitors were only slightly affected, as shown by comparison with the control cells. Interestingly, piceatannol inhibited only partially the activation of Syk (a nonreceptor tyrosine kinase), which has been shown to play a role in a number of endothelial cell functions, including cell growth and migration. These findings suggest that H(+)-ATP synthase-like molecules on the surface of HUVECs play an important role not only in extracellular ATP synthesis but also in the proliferation of HUVECs. The present results demonstrate that the use of small molecular H(+)-ATP synthase inhibitors targeting the F(1) catalytic domain may lead to significant advances in potential antiangiogenic cancer therapies.     

The characterization and regulatory properties of pyruvate kinase from cotton seeds

Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was partially purified from cotton seeds. The enzyme shows normal kinetics toward phosphoenol-pyruvate, ADP, and magnesium or manganese. Of nearly 50 metabolites tested, the enzyme is inhibited only by ATP, UTP, citrate, and malate, and activated by AMP, GMP, and fumarate. The inhibition by citrate and ATP is not due to metal chelation; both compounds appear to directly affect the enzyme. The kinetics of the activations by AMP and by fumarate suggest the existence of separate activator sites for the two compounds.It is suggested that cotton seed pyruvate kinase is a regulatory enzyme, although it differs markedly from the regulatory pyruvate kinases which have been described in animals and in microorganisms. This is the first instance in which regulatory properties have been reported for a pyruvate kinase from a higher plant.     

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.     

Magnetic resonance studies of the interaction of Co2+ and phosphoenolpyruvate with pyruvate kinase.

Co2+, which activates rabbit muscle pyruvate kinase, competes with Mn2+ for the active site of the enzyme with a KD of 46 muM. Co2+ binds to phosphoenolpyruvate with a KD of 4.1 mM. The structures of the binary Co2+/P-enolpyruvate, and quaternary pyruvate kinase/Co2+/K+/P-enolpyruvate complexes were studied using EPR and the effects of Co2+ on the longitudinal (T1) and transverse (T2) relaxation times of the protons of water and P-enolpyruvate and the phosphorus of P-enolpyruvate. The EPR spectra of all complexes at 6 K, disappear above 40 K and reveal principal g values between 2 and 7 indicating high spin Co2+. For free Co2+ and for the binary Co2+/P-enolpyruvate complex, the T1 of water protons was independent of frequency in the range 8, 15, 24.3, 100, and 220 MHz. Assuming coordination numbers (q) of 6 and 5 for free Co2+ and Co2+/P-enolpyruvate, respectively, correlation times (tauc) of 1.3 times 10(-13) and 1.7 times 10(-13) s, were calculated. The distances from Co2+ and phosphorus and to the cis and trans protons in the binary Co2+/P-enolpyruvate complex calculated from their T1 values were 2.7 A, 4.1 A, AND 5.3 A, respectively, indicating an inner sphere phosphoryl complex. Consistent with direct phosphoryl coordination, a large Co2+ to phosphorus hyperfine contact coupling constant (A/h) of 5 times 10(5) Hz was determined by the frequency dependence of the T2 of phosphorus at 25.1, 40.5, and 101.5 MHz. For both enzyme complexes, the dipolar correlation time tauc was 2 times 10(-12) s and the number of rapidly exchanging water ligands (q) was 0.6 as determined from the frequency dependence of the T1 of water protons. In the quaternary enzyme/Co2+/K+/P-enolyruvate complex this tauc value was consistent with the frequency dependence of the T1 of the phosphorus of enzyme-bound P-enolpyruvate at 25.1 and 40.5 MHz. Distances from enzyme-bound C02+ to the phosphorus and protons of P-enolpyruvate, from their T1 values, were 5.0 A and 8 to 10 A, respectively, indicating a predominantly (greater than or equal to 98%) second spere complex and less than 2% inner sphere complex. Consistent with a second sphere complex on the enzyme, an A/h value of less than 10(3) Hz was determined from the frequency dependence of the T2 of phosphorus. In all complexes the exchange reates were found to be faster than the paramagnetic relaxation rates and the hyperfine contact interaction was found to be small compared to the dipolar interaction. The results thus indicate that the interaction of C02+ with P-enolpyruvate is greatly decreased upon binding to the active site of pyruvate kinase.     

Synthesis and biochemical characterization of the new sulfhydryl-reactive ATP analogue 8-thiocyano-ATP. Its interaction with Na,K-ATPase and kinases.

The synthesis of 8-thiocyano-ATP (CNS8-ATP) is described. At 37 degrees C the ATP analogue inactivates Na,K-ATPase, hexokinase, and pyruvate kinase. In all three cases, inactivation can be prevented by the addition of ATP, thus indicating that CNS8-ATP is recognized within the ATP binding site of the above enzymes. Incubation of the inactivated enzymes with dithiothreitol restores the catalytic activities. Therefore, it is likely that in these enzymes a mixed disulfide (E-S-S8-ATP) is formed between a sulfhydryl in the ATP binding site (E-SH) and the ATP analogue: [formula: see text] From the pseudo-first-order inactivation kinetics, a KD = 2.7 microM with k2 = 0.142 min-1 is calculated for the hexokinase and a KD = 40 microM with k2 = 0.347 min-1 is calculated for the pyruvate kinase interactions with the ATP analogue. At 4 degrees C, Na,K-ATPase recognizes CNS8-ATP with a KD = 8.3 microM. At 37 degrees C, the enzyme becomes inactivated by the ATP analogue in a biphasic manner. Inactivation results in the incorporation of [alpha-32P]8-CNS8-ATP into the catalytic alpha-subunit of the enzyme. Limited tryptic digestion in the presence of 150 mM KCl results in the formation of a radioactive peptide of Mr = 56,000, known to bear the purine binding domain of Na,K-ATPase. The results described in this article verify CNS8-ATP as a sulfhydryl-reactive ATP analogue and characterize this new ATP analogue as a useful tool for structure/function studies on ATP-recognizing enzymes.     

Requirement of medium ADP for the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans Fo.F1-ATP synthase

Fo.F1-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes Pi-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP.Mg2+ -induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319-12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 x 10(-6) M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and Pi), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans Fo.F1-ATP synthase.     

Nuclear magnetic resonance studies of substrate interaction with cobalt substituted alcohol dehydrogenase from liver.

The role of zinc in liver alcohol dehydrogenase has been studied by replacement of 1.3 and 3.5 of the four Zn(II) ions with Co(II) and measuring the effects of the paramagnetic Co(II) on the relaxation rates of the protons of water, ethanol, and isobutyramide. Water relaxation studies at 8, 24, 100, and 220 MHz indicate two classes of bound Co(II). The similar to 2 readily replaced Co(II) ions retain one fast exchanging water proton in their inner coordination spheres, while the similar to 2 slowly exchanging Co(II) ions coordinate no detectable water protons, indicating that the former replaced Zn(II) at the "catalytic sites" and the latter replaced Zn(II) at the "structural sites" detected crystallographically. Ethanol, acetaldehyde, and isobutyramide bind with appropriate affinities to the Co(II) substituted alcohol dehydrogenases decreasing the number of fast exchanging protons at the catalytic Co(II) site by greater than or equal to 54 percent. Coenzyme binding causes smaller changes in the water relaxation rate which may be due to local conformation changes. The paramagnetic effects of Co(II) at the catalytic site on the relaxation rates of the methyl protons of isobutyramide at 100 and 220 MHz indicate that this analog binds at a site 9.1 A from the catalytic Co(II). This distance decreases to 6.9 A when NADH is bound, and a Co(II) to methyne proton distance of 6.6 A is determined indicating a conformation change leading to the formation of a second sphere enzyme-Co(II)-isobutyramide complex in which a hydroxyl or water ligand intervenes between the metal and the substrate analog. Similar behavior is observed in the enzyme-ethanol complexes. The paramagnetic effects of Co(II), at the catalytic site, on the relaxation rates of the protons of ethanol at 100 and 220 MHz, indicate that this substrate bind at a site 12-14 A distant from the catalytic Co(II) but that this distancedecreases to 6.3 A in the abortive enzyme-NADH-ethanol complex. The role of the catalytic Co(II) thus appears to be the activation of a hydroxyl or water ligand which polarizes the aldehyde carbonyl group by hydrogen bonding. The role of the structural Co(II), which is more distant from isobutyramide (9-11 A), may be that of a template for protein conformation changes. By combining the present distances with those from previous magnetic resonance studies on the liver enzyme, the arrangement of coenzyme, metal, and substrate at the active site in solution can be constructed. This arrangement is consistent with that of ADP-ribose and zinc in the crystalline complex of liver alcohol dehydrogenase as determined by X-ray crystallography (Branden et al., (1973), Proc. Natl. Acad. Sci. U.S.A.70, 2439).     

NMR studies of the AMP-binding site and mechanism of adenylate kinase

NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S. A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20 degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A, indicating a reaction coordinate distance of approximately 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (less than or equal to 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y., & Nakazawa, A. (1986) J. Biol. Chem. 261, 2942-2945].