Structural and biochemical properties of bidentate tetraaquarhodium(III) complexes of inorganic pyrophosphate and adenosine 5'-diphosphate

The structural and biochemical properties of the alpha,beta-bidentate tetraaquarhodium(III) complexes of inorganic pyrophosphate [Rh(H2O)4PP] and adenosine diphosphate [Rh(H2O)4ADP] are examined. These Rh(III) complexes are exchange-inert analogues of the corresponding physiologically important MgIIPP and MgIIADP complexes. The crystal structure of [Rh(H2O)4H2P2O7]+Cl- shows that the six-membered chelate ring adopts a twist-boat conformation with an unusually high puckering amplitude of 0.756 (3) A. The Rh coordination distances average 2.02 (1) A, while the bridge P-O bonds are virtually equal in length. All 10 protons of the complex participate in hydrogen bonding. There are two intramolecular hydrogen bonds between the phosphate oxygen atoms and the axially coordinated water molecules. The Rh(H2O)4PP complex was found to be a substrate for yeast inorganic pyrophosphatase, with Ki = 0.063 (7) mM and Vm = 500 (100) min-1. The two screw sense isomers of Rh(H2O)4ADP were prepared from (Rp)-[alpha-16O,18O]ADP and assigned configuration on the basis of the magnitude of their 31P NMR isotopic chemical shifts. The Rh(H2O)4ADP complex binds a number of kinases as tightly as MgADP. Arginine kinase and creatine kinase were shown to bind the delta Rh(H2O)4ADP isomer 7 and 45 times tighter, respectively, than the lambda isomer. The reactivity of Rh(H2O)4PP with pyrophosphatase is comparable to that of Cr(H2O)4PP, and the binding affinities of the Rh(H2O)4ADP screw sense isomers for kinases are also comparable to those observed for the corresponding Cr(H2O)4ADP screw sense isomers.     

Triamminemonoaquocobalt(III) complexes of pyrophosphate and adenosine diphosphate (ADP)

Cobalt(III)H2O(NH3)3 pyrophosphate has been shown by proton and 31P nuclear magnetic resonance (NMR) to be a facial bidentate complex. Cobalt(III)H2O(NH3)3 adenosine diphosphate has been resolved into lambda and delta isomers by chromatography on cycloheptaamylose. Both the lambda and delta forms are a pair of isomers that are not separated by cycloheptaamylose, reverse phase high-pressure liquid chromatography (HPLC), or cation exchange chromatography. These isomers presumably represent syn- and anti-arrangement of coordinated water and adenosine.     

How do MgATP analogues differentially modify high-affinity and low-affinity ATP binding sites of Na+/K(+)-ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.     

Inactivation of yeast phosphoglycerate kinase by Cr-ATP complexes and its implications on the conformation of the enzyme active site.

Exchange-inert beta, gamma-bidentate Cr(H2O)x(NH3)y ATP complexes inactivate yeast phosphoglycerate kinase (PGK) by forming a coordination complex at the enzyme active site. The observed inactivation rates ranged from 0.019 min-1 to 0.118 min-1 for Cr(NH3)4ATP and Cr(H2O)4ATP, respectively. Incorporation of one mol of Cr-ATP to the enzyme was sufficient for complete inactivation of the enzyme. The presence of Mg-ATP protected the enzyme against inactivation by Cr-ATP. The other substrate 3-phosphoglycerate (3-PGA), when present, reduced the observed inactivation rates. The reduction of the k(obs) by 3-PGA was proportional to the number of NH3 ligands present in the coordination sphere of Cr3+ in the Cr-ATP complex, suggesting that in the ternary enzyme-Cr-ATP-3-PGA complex 3-PGA may be coordinated to the metal ion. When the effector sulfate ion was present, the presence of 3-PGA did not cause any further effects on the observed inactivation rates. This suggests that bound substrates are in a different arrangement at the active site when sulfate is present and therefore 3-PGA may not need to displace a ligand from Cr3+. Additionally, PGK exhibited a stereoselectivity for the binding of Cr(H2O)4ATP. delta diastereomer of Cr(H2O)4ATP yielded an order of magnitude smaller Ki value compared to the value observed with the lambda isomer. The recovery of enzyme activity was observed over a period of a few hours upon removal of excess Cr-ATP. The presence of substrates and/or effector ion sulfate did not alter the observed reactivation rate. There was no difference in the reactivation rates of the enzyme which was inactivated with Cr(H2O)4ATP or Cr(NH3)4ATP with and without 3-PGA. Increasing the ligand exchange rates of Cr3+ of Cr-ATP by increasing the pH value of the recovery medium from 5.9 to 6.8 increased the rate of recovery by a factor of 8. The pH dependence of the reactivation indicated that one hydroxyl group is involved in the recovery of the enzyme activity in enzyme CrATP and enzyme.CrATP.3-PGA complexes.     

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.     

Investigation of substrate specificity of creatine kinase using chromium (III) and cobalt(III) complexes of adenosine 5'-diphosphate

The specificity for substrate binding to creatine kinase for metal-nucleotide complexes of the type Cr-(H2O)4-n(NH3)nADP (where n = 0, 3, or 4) and Co-(H2O)4-m(NH3)mADP (for m = 3 or 4) has been investigated over the pH range 5.5-7.8 with the delta-alpha, beta-bidentate diastereoisomers. These inert nucleotide complexes acted as competitive inhibitors vs. MgADP over this range. In addition, the pH dependence of the V, V/K, and Km values for MgADP has been determined. Metal-nucleotide binding to the enzyme is strongest below an approximate pK of 6.45 but again becomes pH independent above pH 7. This pK is not associated with the metal-nucleotide complex. Instead, we conclude that the pK of the acid-base catalyst (thought to be histidine) is about 6.45 in the absence of nucleotide but is raised to 7.2 in its presence. This perturbation of the pK may result from a protein conformational change that allows a hydrogen bond to form between the phosphorylated nitrogen of phosphocreatine and the acid-base catalyst. The pK of the water in Cr(H2O)(NH3)3ADP has been determined to be 6.6, and by comparison of the binding affinity of this complex with that of Cr(NH3)4ADP or Cr(H2O)4ADP, it can be deduced that the hydroxo species binds more strongly than the aquo complex. In general, chromium nucleotides are bound more strongly than cobalt complexes, and binding affinity increases as water replaces ammonia in the first coordination sphere of the metal. Both trends are a result of stronger hydrogen-bond interactions between the metal complex and protein.     

Interaction of purine nucleotides with inert paramagnetic Cr(III) probes evaluated by NMR relaxation effects. Molecular mechanics calculations on Cr(III) and Co(III) polyphosphate complexes

The 1H NMR relaxation effects produced by paramagnetic Cr(III) complexes on nucleoside 5'-mono- and -triphosphates in D2O solution at pH' = 3 were measured. The paramagnetic probes were [Cr(III)(H2O)6]3+, [Cr(III)(H2O)3(HATP)], [Cr(III)(H2O)3(HCTP)] and [Cr(III)(H2O)3(UTP)-, while the matrix nucleotides (0.1 M) were H2AMP, HIMP-, and H2ATP2-. For the aromatic base protons, the ratios of the transverse to longitudinal paramagnetic relaxation rates (R2p/R1p) for the [Cr(III)(H2O)6]3+/H2ATP2-, [Cr(III)(H2O)3(HATP)]/H2ATP2-, [Cr(III)(H2O)3(HCTP)]/H2ATP2 and [Cr(III)(H2O)3(UTP)]-/H2ATP2 systems were below 2.33 so the dipolar term predominates. For a given nucleotide, R1p for the purine H(8) signal was larger than for the H(2) signal with the [Cr(III)(H2O)6]3+ probe, while R1p for the H(2) signal was larger with all the other Cr(III) probes. Molecular mechanics computations on the [Cr(III)(H2O)4(HPP)(alpha,beta)], [Cr(III)(NH3)4(HPP)(alpha,beta)], [Co(III)(NH3)3(H2PPP)(alpha,beta,gamma)] and [Co(III)(NH3)4(HPP)(alpha,beta)] complexes gave calculated energy-minimized geometries in good agreement with those reported in crystal structures. The molecular mechanics force constants found were then used to calculate the geometry of the inner sphere [Cr(III)(H2O)6]3+ and [Cr(III)(H2O)3(HATP)(alpha,beta,gamma)] complexes as well as the structures of the outer sphere [Cr(III)(H2O)6]3(+)-(H2AMP) and [Cr(III)(H2O)6]-(HIMP)- species. The gas-phase structure of the [Cr(III)(H2O)3(HATP)(alpha,beta,gamma)] complex shows the existence of a hydrogen bond interaction between a water ligand and the adenine N(7)(O...N = 2.82 A). The structure is also stabilized by intramolecular hydrogen bonds involving the -O(2')H group and the adenine N(3) (O...N = 2.80 A) as well as phosphate oxygen atoms and a water molecule (O...O = 2.47 A). The metal center has an almost regular octahedral coordination geometry. The structures of the two outer-sphere species reveal that the phosphate group interacts strongly with the hexa-aquochromium probe. In both complexes, the nucleotides have a similar "anti" conformation around the N(9)-C(1') glycosidic bond. However, a very important difference characterizes the two structures. For the (HIMP)- complex, strong hydrogen bond interactions exist between one and two water ligands and the inosine N(7) and O(6) atoms, respectively (O...O = 2.63 A; O...N = 2.72, 2.70 A). For the H2AMP complex, the [Cr(III)(H2O)6]3+ cation does not interact with N(7) since it is far from the purine system. Hydrogen bonds occur between water ligands and phosphate oxygens. The Cr-H(8) and Cr-H(2) distances revealed by the energy-minimized geometries for the two outer sphere species were used to calculate the R1p values for the H(8) and H(2) signals for comparison with the observed R1p values: 0.92(c), 1.04(ob) (H(8)) and 0.06(c), 0.35(ob) (H(2)) for H2AMP; and 3.76(c), 4.53(ob) (H(8)) and 0.16(c), 0.77(ob) s-1 (H(2)) for HIMP-.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chromium(III) beta, gamma-bidentate guanine nucleotide complexes as probes of the GTP-activated cGMP cascade of retinal rod outer segments

The exchange-inert Cr(III) beta, gamma-bidentate guanine nucleotide complexes Cr(III)GTP and Cr(III)Gpp(NH)p were used to probe the role of transducin in activating the retinal cGMP cascade. The Cr(III) nucleotide complexes were found to have lower binding affinity for transducin as compared to the Mg2+ complexes. However, the rate of hydrolysis of the transducin-bound Cr(III)GTP was similar to that of Mg(II)GTP. Cr(III)Gpp(NH)p activated the cGMP phosphodiesterase of photolyzed rod outer segment membranes up to 75% of the Mg(II)Gpp(NH)p level but lacked the ability to dissociated the transducin subunits from the rod outer segment membrane. This result implies that the activation of the phosphodiesterase by transducin-GTP complex is a membrane-associated event and the formation of a soluble complex of transducin-GTP with the inhibitory peptide of the phosphodiesterase may not be an obligatory step. Both the delta and lambda screw sense stereoisomers of Cr(III)Gpp(NH)p were capable of activating the cGMP cascade with no apparent stereoselectivity. The nature of the interaction of the metal ion and GTP at the nucleotide-binding site of transducin is discussed together with the results from previous studies using the phosphorothioate GTP analogues [Yamanaka, G., Eckstein, F., & Stryer, L. (1985) Biochemistry 24, 8094-8101] and is compared to the site found in homologous GTP-binding proteins such as elongation factor Tu [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36; la Cour, T.F.M., Nyborg, J., Thirup, S., & Clark, B.F.C. (1985) EMBO J. 4, 2385-2388]. The implications of the observed results on the molecular mechanism of visual signal transduction are discussed.     

Investigation of the regiospecificity and stereospecificity of proton transfer in the yeast inorganic pyrophosphatase catalyzed reaction

The regiospecificity and stereospecificity of proton transfer in the yeast inorganic pyrophosphatase (PPase) catalyzed hydrolysis of P1,P2-bidentate Mg(H2O)4(PPi)2- were probed with exchange-inert metal complexes of imidodiphosphate (PNP) and thiopyrophosphate (PPS). PPase was unable to catalyze the hydrolysis of Mg(H2O)4PNP and P1,P2-bidentate Co(NH3)4PNP under conditions that resulted in rapid hydrolysis of the corresponding metal-PPi complexes. PPase was found to catalyze the hydrolysis of Mg(H2O)4PPS at 17% the rate of Mg(H2O)4PPi hydrolysis. The Km of Mg(H2O)4PPS was determined to be 300 microM, which is a value 10-fold greater than that observed for Mg(H2O)4PPi. P1,P2-Bidentate Cr(H2O)4PPS and Co(NH3)4PPS (prepared from PPS) were both found to be substrates for PPase. The enzyme specifically catalyzed the hydrolysis of the Rp enantiomers of these complexes and not the Sp enantiomers. These results are accommodated by a reaction mechanism involving enzyme-mediated proton transfer to the pro-R oxygen atom of the incipient phosphoryl leaving group of the bound P1,P2-bidentate Mg(H2O)4PPi2- complex.     

Purification and molecular characteristics of mitochondrial phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver

Phosphoenolpyruvate carboxykinase from bullfrog liver mitochondria has been purified to electrophoretical and immunological homogeneity by an improved method using hydrophobic chromatography on Sepharose-hexane-GMP and affinity chromatography on phosphocellulose. The molecular weight was determined to be 70,000 by SDS-gel electrophoresis, 65,000 by Sephadex G-100 gel filtration and 72,000 by glycerol gradient centrifugation. The isoelectric point was determined to be 6.2, differing from that of the cytosol enzyme. The rabbit IgG fraction against the mitochondrial PEP carboxykinase precipitated not only the mitochondrial but also the cytosol enzyme. The dissociation constant of the nucleotide-enzyme complex was determined to be 3 microM for GTP, 8.5 microM for GDP, and 171 microM for GMP. The affinity of GTP for the enzyme was reduced in the presence of phosphoenolpyruvate or Mn2+, whereas that of GDP was not changed. GMP inhibited the enzyme competitively with GDP for the phosphoenolpyruvate carboxylation and competitively with GTP for the exchange reaction between [14C]HCO3- and oxaloacetate. The purified enzyme was found to have a cysteine residue which reacted with iodoacetamide to form inactive enzyme. Guanine nucleotides or IDP and Mn2+ at a lower concentration prevented the inactivation by iodoacetamide of the enzyme in a competitive manner. Binding of guanine nucleotide to the enzyme and the relation of the sulfhydryl group to the nucleotide binding are discussed.     

Neutral complexes as oxidants for the reduced form of parsley (Petroselinum crispum) [2Fe--2S] ferredoxin. Evidence for partial blocking by redox-inactive Cr(III) complexes.

The 1 : 1 reactions of three neutral Co(III) oxidants, Co(acac)3, Co(NH3)3(NO2)3 and Co(acac)2(NH3)(NO2), with reduced parsley (Petroselinum crispum) [2Fe--2S] ferredoxin (which carries a substantial negative charge), have been studied at 25 degrees C, pH 8.0 (Tris/HCl), I0.10 (NaCl). Whereas it has previously been demonstrated that with Co(NH3)6+ as oxidant the reaction if completely blocked by redox-inactive Cr(NH3)63+, the neutral oxidants are only partially blocked by this same complex. The effects of three Cr(III) complexes, Cr(NH3)63+%, Cr(en)33+ and (en)2Cr . mu(OH,O2CCH3) . CR(en)24+ have been investigated. Kinetic data for the response of 3+, neutral, as well as 1--oxidants to the presence of 3+ (and 4+) Cr(III) complexes can now be rationalized in terms of a single functional site on the protein for electron transfer. Electrostatics have a significant influence on association at this site.     

Stimulation of human fat cell adenylate cyclase by GDP and guanosine 5'-O-(2-thiodiphosphate)

GDP regulation of basal and receptor-mediated catecholamine-sensitive human fat cell adenylate cyclase was studied using purified plasma membrane preparations and assay conditions selected to minimize conversion of GDP to GTP. Under ordinary assay conditions (low NaCl concentration) and with App(NH)p as substrate to prevent GDP conversion to GTP, basal enzyme activity was stimulated up to 2-fold by GDP (0.1 mM) while addition of epinephrine (0.1 mM) eliminated stimulation by GDP and reduced basal adenylate cyclase activity. With ATP as substrate, the enzyme was not responsive to hormone in the absence of guanyl nucleotides and GDP augmentation of basal activity was small (0-1.5-fold) while stimulatory effects of epinephrine and isoproterenol were minimally but definitely exhibited (1.5-fold over basal). Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a GDP analog resistant to phosphorylation and hydrolysis and an antagonist of GTP, stimulated enzyme activity more than did GDP but did not promote epinephrine action. Rather, inhibition of GDP beta S-stimulated adenylate cyclase activity was seen with both epinephrine and isoproterenol and also with GTP. In the presence of NaCl (200 mM), which alone produced 2-3-fold increase in basal enzyme activity, GDP (0.1 mM) and GDP beta S (50 microM) produced 8- and 15-fold increases of activity, respectively. Addition of UDP, to prevent possible conversion of GDP to GTP, had no effect on NaCl-enhanced activation by GDP. The results indicate that the human fat cell adenylate cyclase system is unique in responding to GDP and its analog GDP beta S by stimulation in the absence of hormone but suggest that as in other systems catecholamine-mediated stimulation is normally dependent on GTP. Salts (Na+) appear to stimulate the enzyme by facilitating the interaction of the guanyl nucleotide regulatory protein (N8) with the catalytic unit.     

Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP.

The interaction of sarcoplasmic reticulum Ca(2+)-ATPase with the Mg.ATP analogues Rh(H2O)4ATP and Co(NH3)4ATP have been examined. Co(NH3)4ATP slowly inactivates Ca(2+)-ATPase in a first order process, with a rate constant of 1.13 x 10(-3) s-1 and an apparent inactivation constant, KI, of 32 mM. Rh(H2O)4ATP likewise inactivates sarcoplasmic reticulum Ca(2+)-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H2O)4ATP] is biphasic. The chi-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca(2+)-ATPase by Rh(H2O)4ATP of KI1 = 30 microM and KI2 = 221 microM. The corresponding values of k2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 x 10(-4)s-1. Tridentate Rh(H2O)3ATP also inhibits Ca(2+)-ATPase, but only after much longer incubation times. Ca(2+)-ATPase inactivation is accompanied by incorporation of radioactivity from gamma-32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca(2+)-dependent ATPase activity in this experiment was 2.19 x 10(-4)s-1, and the first-order rate constant for Ca(2+)-dependent E-P formation was 2.07 x 10(-4)s-1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca(2+)-ATPase by Rh(H2O)4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H2O)4ATP is binding to the catalytic nucleotide site on Ca(2+)-ATPase and producing a highly stable, phosphorylated intermediate.     

Inhibition and inactivation of pyruvate phosphate dikinase with Cr(III) complexes of adenosine 5'-triphosphate and inorganic pyrophosphate

The exchange inert complexes beta,gamma-bidentate Cr(H2O)4ATP and P1,P2-bidentate Cr(H2O)4PP were found to bind to the Bacteriodes symbiosus pyruvate phosphate dikinase ATP and PP binding sites, respectively. The inactivation of the enzyme that was observed with these complexes was shown to involve covalent attachment of the entire complex to the enzyme via insertion of enzyme amino acid side chains into the coordination sphere of the Cr(III). Incubation of Cr(H2O)4ATP with other proteins also resulted in covalent attachment.     

Guanosine thiophosphate derivatives as substrate analogues for phosphoenolpyruvate carboxykinase

The interactions of nucleotides with phosphoenolpyruvate carboxykinase were studied by using the stereospecific thiophosphate analogues of GDP and GTP. The metal ion dependent stereoselectivity of these analogues was determined by using steady-state kinetics. The RP and SP isomers of guanosine 5'-O-(1-thiodiphosphate) (GDP alpha S) were substrates with low turnover, and a small preference for the RP isomer was observed. Neither the enzyme-metal nor the nucleotide-metal complex elicited any substantial change in the selectivity. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) exhibited no substrate activity for the enzyme, regardless of the cations. This nucleotide was a competitive inhibitor against GDP, however. Both RP and SP diastereomers of guanosine 5'-O-(1-thiotriphosphate) (GTP alpha S) were good substrates for phosphoenolpyruvate carboxykinase; in several cases, depending upon the cation, kcat and/or Vm/Km for the RP isomer is greater than for the substrate GTP. The enzyme-metal complex but not the nucleotide-metal complex affects the relative Km and the Vmax values. In contrast, guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) (SP) is a much better substrate (greater than 50 times) than is GTP beta S (RP). The metal ions have little effect on the selectivity. These results suggest a specific interaction of the beta-phosphate of the nucleotide with the protein. The analogue guanosine 5'-O-(3-thiotriphosphate) (GPT gamma S) serves as a substrate to yield GDP and thiophosphoenolpyruvate. The latter was detected by 31P NMR and was shown to slowly hydrolyze to form phosphoenolpyruvate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigations of the metal ion-binding sites of yeast inorganic pyrophosphatase

Yeast inorganic pyrophosphatase was found to bind two Mn2+ per subunit in the absence of phosphate and three Mn2+ per subunit in the presence of phosphate. Kinetic studies of the pyrophosphatase-catalyzed hydrolysis of Cr(NH3)4PP and Cr(H2O)4PP were carried out with Mn2+ and with Mg2+ as activators. The results from these studies suggest that three divalent cations per pyrophosphatase active site are required for catalysis. NMR and EPR studies were conducted to evaluate the relative location of the metal ion binding sites on the enzyme. The two Mn2+ ions bound to the free enzyme are in close enough proximity to magnetically interact. Analysis of the NMR and EPR data in terms of a dipolar relaxation mechanism between Mn2+ ions provides an estimate of the distance between them of 10-14 A. When the diamagnetic substrate analog [Co(NH3)4PNP]- or intermediate analog [Co(NH3)4 (P)2]- are bound to pyrophosphatase, two Mn2+ ions still bind to the enzyme and their magnetic interaction increases. In the presence of these Co3+ complexes, the Mn2+--Mn2+ separation decreases to 7-9 A. Several NMR and EPR experiments were conducted at low Mn2+ to pyrophosphatase ratios (approximately 0.3), where only one Mn2+ ion binds per subunit, in the presence of Cr3+ or Co3+ complexes of PNP or PP. Analysis of the Mn2+--Cr3+ dipolar relaxation evident in proton NMR and EPR data provided for the calculation of Mn2+--Cr3+ distances. When the substrate analog CrPNP was present, the Mn2+--Cr3+ distance was congruent to 7 A whereas, when Cr(P)2 was bound to pyrophosphatase, the Mn2+--Cr3+ distance was congruent to 5 A. These results strongly support a model for the catalytic site of pyrophosphatase that involves three metal ion cofactors.     

Cytotoxicity studies of chromium(III) complexes on human dermal fibroblasts

The cytotoxicity of certain Cr(III) complexes, such as [Cr(salen)(H(2)O)(2)](+), [Cr(edta)(H(2)O)](-), [Cr(en)(3)](3+), [Cr(ox)(3)](3-), [Cr(pic)(3)], and CrCl(3), which differ in ionic character and ligand environment in human dermal skin fibroblasts, has been studied. After 72 h of exposure to 100 microM doses of chromium(III) complexes, the order in which the complexes had an inhibitory effect on cell viability was [Cr(en)(3)](3+) > [Cr(salen)(H(2)O)(2)](+) > [Cr(ox)(3)](3-) > [Cr(edta)(H(2)O)](-) > [Cr(pic)(3)] > CrCl(3). Based on viability studies it was confirmed that [Cr(en)(3)](3+), a triply charged cation, inhibits cell proliferation, and therefore, it was chosen to carry out further investigations. [Cr(en)(3)](3+), at a dose of 50 microM, was found to bring about surface morphological changes, evidenced by cellular blebbing and spike formation accompanied by nuclear damage. TEM analysis revealed substantial intracellular damage to fibroblasts in terms of the formation of apoptotic bodies and chromatin condensation, thus reflecting cell death. FACS analysis further revealed DNA damage by formation of a sub-G(1) peak with 84.2% DNA as aneuploid DNA and arrest of the G(2) / M phase of the cell cycle. Cellular DNA damage was confirmed by agarose gel electrophoresis with the characteristic appearance of a DNA streak in DNA isolated from [Cr(en)(3)](3+)-treated fibroblasts. The proposed mechanism suggests the plausible role of Cr(V), formed as a result of oxidation of Cr(III) by cellular oxidative enzymes, in the cytotoxic response. Consequently, any Cr(III) complex that is absorbed by cells and can be oxidized to Cr(V) must be considered a potential carcinogen. This has potential implications for the increased use of Cr(III) complexes as dietary supplements and highlights the need to consider the cytotoxicity and genotoxicity of a variety of Cr(III) complexes and to understand the potential hazards of Cr(III) complexes encountered in research laboratories.     

Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactin-producing rat pituitary tumor cells (GH cells)

A thyroliberin (TRH)-responsive particulate bound adenylyl cyclase is present in two rat anterior pituitary tumor cell strains (GH4C1 and GH3) which synthesize and secrete prolactin. At a given Mg2+ concentration, ATP and the guanyl nucleotides GTP and guanyl 5'-yl-imidodiphosphate (GMP-P(NH)P) caused a dose-dependent increase in adenylyl cyclase activity. The maximum response to thyroliberin occurred with ATP and GTP at concentrations above 0.30 mM and 2 microM, respectively. The maximal stimulatory effect of thyroliberin on adenylyl cyclase activity was 2-fold in the presence of GTP. GMP-P(NH)P increased the basal enzyme activity 4- to 10-fold over and above that of equimolar concentrations of GTP but supported poorly the TRH-induced response. Mg2+ caused a dose-dependent increase in the basal enzyme activity and reduced TRH and fluoride-induced responses. Also, Mn2+ and Co2+ stimulated the basal adenylyl cyclase activity while Zn2+, Ca2+, and Cu2+ inhibited the enzyme, and neither cations supported the TRH response. Half-maximal stimulation of the adenylyl cyclase by TRH and half-maximum binding of [3H]TRH to membranes at 35 degrees C were 102 and 56 nM, respectively. Pretreatment with TRH decreased the apparent Vmax of the enzyme and the maximal binding of [3H]TRH. Of 6 TRH analogs tested, only one was able to displace [3H]TRH from its receptor and to increase the adenylyl cyclase activity. We suggest that adenylyl cyclase activation is an early event in the stimulus secretion coupling between TRH and prolactin-producing GH cells.     

Effects of Mn2+ on the exchange reaction of phosphoenolpyruvate carboxykinase in the presence of high concentrations of Mg2+

The mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn2+ and Mg2+ in the oxaloacetate ---- H14CO-3 exchange reaction. Increases in the Mg2+ concentration caused decreases in the K0.5 value of Mn2+ in line with the earlier finding that the enzyme was markedly activated by low Mn2+ (microM) plus high Mg2+ (mM). In the presence of 2.5 mM Mg2+, increases in the Mn2+ level first enhanced the activity of phosphoenolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn2+ alone. Kinetic studies showed that high Mn2+ inhibited the activity of Mg2+ noncompetitively, and those of GTP and oxaloacetate uncompetitively. The inhibition constant for oxaloacetate (K'i = 550 microM) was lower than that of Mg2+ (Ki = K'i = 860 microM) or GTP (K'i = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn2+. These results suggested that Mn2+ can play two roles, of activating and suppressing phosphoenolpyruvate carboxykinase activity in the presence of high Mg2+.     

Kinetic mechanism of phosphoenolpyruvate carboxykinase (GTP) from rat liver cytosol. Product inhibition, isotope exchange at equilibrium, and partial reactions

Initial velocity studies of rat liver cytosolic P-enolpyruvate carboxykinase in the direction of P-enolpyruvate formation gave intersecting double reciprocal plots indicating that the reaction conforms to a sequential reaction pathway. A complete product inhibition study with MnGDP-, P-enolpyruvate, and HCO3- as product inhibitors indicated that all patterns were noncompetitive. Isotope exchange at equilibrium with exchange between the substrate/product pairs GTP/GDP oxalacetate/HCO3-, and oxalacetate/P-enolpyruvate while varying the concentration of substrate/product pairs in fixed constant ratio gave no complete inhibitory patterns as the concentration of the constant ratio pairs approached saturation. The exchange rates between the substrate/product pairs differed by a factor of 40 when compared under the same assay conditions. These results were interpreted in terms of a random reaction mechanism in which true dead-end complexes do not form and in which the rate-limiting step is not the interconversion of the ternary quarternary central complexes. In addition to the formation of P-enolpyruvate from oxalacetate and MnGTP2-, the enzyme catalyzes the decarboxylation of oxalacetate to pyruvate in the absence of MnGTP2-. This reaction occurs only slowly in the absence of GDP and most rapidly in the presence of MnGDP-. When only MnGTP2- and oxalacetate are present, no pyruvate is formed, and oxalacetate is converted stoichiometrically to P-enolpyruvate. The enzyme also catalyzes the exchange of [14C]GDP into GTP in the absence of P-enolpyruvate. This exchange is stimulated by the presence of HCO3-. When enzyme is incubated with MnGTP2- in the presence or absence of HCO3-, there is no hydrolysis to form GDP and P1. The two partial reactions, namely the exchange of [14C]GDP with the E.HCO3.MnGTP or E.MnGTP complex and the formation of pyruvate from the E.oxalacetate.MnGDP complex provide pathways by which the expected dead-end complexes can be converted to enzyme forms which can return to the catalytic or exchange sequence.