Magnetic resonance and kinetic studies of the role of the divalent cation activator of RNA polymerase from Escherichia coli.

The interaction of Mn2+, substrates and initiators with RNA polymerase have been studied by kinetic and magnetic resonance methods. As determined by electron paramagnetic resonance, Mn2+ binds to RNA polymerase at one tight binding site with a dissociation constant less than 10 muM and at 6 +/- 1 weak binding sites with dissociation constants 100-fold greater. The binding of Mn2+ to RNA polymerase at both types of sites causes an order of magnitude enhancement of the paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons, indicating the presence of residual water ligands on the enzyme-bound Mn2+. A kinetic analysis of the Mn2+-activated enzyme with poly(dT) as template indicates the substrate to be MnATP under steady-state conditions in the presence or absence of the initiator ApA. ATP and UTP interact with the tightly bound Mn2+ to form ternary complexes with approximately 50% greater enhancement factors. The dissociation constant of MnATP from the tight Mn2+ site as determined by longitudinal proton relaxation rate (PRR) titration (4.7 muM) is similar to the KM of MnATP in the ApA-initiated RNA polymerase reaction (10 +/- 3 muM) but not in the ATP-initiated reaction (160 +/- 30 muM). Similarly, the dissociation constant of the substrate MnUTP from the tight Mn2+ site (90 muM) is in agreement with the KM of MnUTP (101 +/- 13 muM) when poly[d(A-T)]-poly[d(A-T)] is used as template, indicating the tight Mn2+ site to be the catalytic site for RNA chain elongation. Manganese adenylyl imidodiphosphate (MnAMP-PNP) has been found to be a substrate for RNA polymerase. It has the same affinity as MnATP for the tight site but, unlike the results obtained with MnATP, the enhancement is decreased by 43% in the enzyme Mn-AMP-PNP complex. These results suggest that the enzyme-bound Mn2+ interacts with the leaving pyrophosphate group. The initiators ApA and ApU and the inhibitor rifamycin interact with the enzyme-Mn2+ complex producing small (15-20%) decreases in the enhancement. The dissociation constant of ApA estimated from PRR data (less than or equal to 1.5 muM) agrees with that determined kinetically (1.0 +/- 0.5 muM) as the concentration of ApA required to produce half-maximal change in the KM of MnATP. In the presence of the initiation specific reagents ApA, ApU, or rifamycin, the affinity of the enzyme-Mn complex for ATP or UTP shows little change. However, ATP and UTP no longer increase the enhancement factor of the tightly bound Mn2+ but decrease it by 30-55%, indicating a change in the environment of the Mn2+-substrate complex on the enzyme when the initiation site is either occupied or blocked. Although the role of the six weak Mn2+ binding sites is not clear, the presence of a single tightly bound Mn2+ at the catalytic site for chain elongation which interacts with the substrate reinforces the number of active sites as one per molecule of holoenzyme and provides a paramagnetic reference point for further structural studies.     

Magnetic resonance studies on manganese-nucleotide complexes of phosphoglycerate kinase.

Measurements of the relaxation rate of water protons (PRR) have been used to study the interaction of yeast phosphoglycerate kinase with the manganous complexes of a number of nucleotides. The results indicate that phosphoglycerate kinase belongs to the same class of enzymes as creatine kinase, adenylate kinase, formyltetrahydrofolate synthetase, and arginine kinase, with maximal binding of metal ion to tne enzyme in the presence of the nucleotide substrate. However, an analysis of titration curves for a number of nucleoside diphosphates (ADP, IDP, GDP) showed that there is a substantial synergism in binding of the metal ion and nucleotide to the enzyme in the ternary complex. The metal-substrate binds to the enzyme approximately two orders of magnitude more tightly than the free nucleotide; Other evidence for an atypical binding scheme for Mn(II)-nucleoside diphosphates was obtained by electron paramagnetic resonance (EPR) studies; the EPR spectrum for the bound Mn(II) in the enzyme-MnADP complex differed substantially from those obtained for other kinases. An identical EPR spectrum is observed with the MnADP complex with the rabbit muscle enzyme as with the yeast enzyme. In contrast, the dissociation constant for the enzyme-MnATP complex is approximately fourfold lower than that for enzyme-ATP, and there are no substantial changes in the electron paramagnetic resonance spectrum of MnATP2- when the complex is bound to phosphoglycerate kinase. A small but significant change in the PRR of water is observed on addition of 3-phosphoglycerate (but not 2-phosphoglycerate) to the MnADP-enzyme complex. However, addition of 3-phosphoglycerate to enzyme-MnADP did not influence the EPR spectrum of the enzyme-bound Mn(II).     

Involvement of a divalent cation in the binding of fructose 6-phosphate to Trypanosoma cruzi phosphofructokinase: kinetic and magnetic resonance studies

When Mg2+ ions were replaced by Mn2+ in the assay of Trypanosoma (Schizotrypanum) cruzi phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) the Km for D-fructose 6-phosphate (F6P) was reduced threefold while the corresponding constant for ATP was essentially unaffected. A detailed kinetic investigation showed that the apparent Km for F6P decreased monotonically with increasing free Mn2+ concentrations, from a limiting value of 5.7 mM in its absence to a limiting value of 1.1 mM in the presence of saturating concentrations of the ion; the Vmax of the enzyme was, on the other hand, not affected by the concentration of Mn2+. Conversely, it was shown that the apparent Km for Mn2+ at fixed MnATP concentrations decreased with increasing F6P concentrations, from a limiting value of 30 microM in the absence of the sugar phosphate to 9 microM at saturating concentrations of the substrate, while the apparent Vmax increased monotonically from zero to its limiting value. Both electron paramagnetic resonance and water proton longitudinal relaxation studies showed binding of one Mn2+ ion per 18,000 Da catalytic subunit of enzyme in the absence of F6P, with a dissociation constant of 57 +/- 4 microM, comparable to the apparent Km for the ion in the absence of F6P. The presence of saturating level of F6P decreases the value of the dissociation constant of Mn2+ to a limiting value of 7.9 microM in agreement with the results of the kinetic analysis. The substrate F6P decreases the enhancement of the water proton longitudinal relaxation rate in a saturable fashion, suggesting displacement of water molecules coordinated to the enzyme-bound Mn2+ ion by the sugar phosphate. Computer fitting of the several dissociation constants and relaxation enhancements for binary and ternary complexes gives a value of 7.9 mM for the dissociation constant of the enzyme-F6P complex in the absence of Mn2+ and 1.1 mM in the presence of saturating concentrations of the ion, in excellent agreement with the respective Km values of F6P extrapolated to zero and saturating Mn2+, respectively. Studies of the frequency dependence of the water proton longitudinal relaxation rate enhancements in the presence of both binary (enzyme-Mn2+) and ternary (enzyme-Mn2(+)-F6P) complexes, are most simply explained by assuming two exchangeable water molecules in the coordination sphere of the enzyme-bound Mn2+ in the binary complex, while in the ternary complex the data are consistent with the displacement of one of the water molecule from the coordination sphere with no significant alteration of the correlation time. Overall, the kinetic and binding data are consistent with the formation of an enzyme-metal-F6P bridge complex at the active site of T. cruzi phosphofructokinase, a coordination scheme which is unique among the phosphofructokinases.     

Structure of metal-nucleotide complexes bound to creatine kinase: 31P NMR measurements using Mn(II) and Co(II)

The structures of metal-nucleotide complexes bound to rabbit muscle creatine kinase have been studied by making measurements of paramagnetic effects of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), on the spin-relaxation rates of the 31P nuclei of ATP and ADP in these complexes. The experiments were performed on enzyme-bound complexes, thereby limiting the contributions to the observed relaxation rate to two exchanging complexes (with and without the cation). Measurements were made as a function of temperature in the range 5-35 degrees C and at three 31P NMR frequencies, 81, 121.5, and 190.2 MHz, in order to determine the effect of exchange on the observed relaxation rates. The relaxation rates in E X MnADP and E X MnATP are independent of frequency, and their temperature variation yields activation energies (delta E) in the range 5-8 kcal/mol; in the transition-state analogue complex E X MnADP X NO3- X Cre (Cre is creatine), delta E is increased to 17.3 kcal/mol. These results demonstrate that the relaxation rates in the Mn(II) complexes are exchange limited and are incapable of providing structural data. It is shown further that use of line-width measurements to estimate the lifetime of the paramagnetic complex leads to incorrect results. The relaxation rates in E X CoADP and E X CoATP exhibit frequency dependence and delta E values in the range 1-3 kcal/mol; i.e., these rates depend on the Co(II)-31P distances, whereas those in the E X CoADP X NO3- X Cre complex have delta E approximately 18 kcal/mol and are significantly contributed by exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.     

Interactions of phospho- and dephosphosuccinyl coenzyme A synthetase with manganous ion and substrates. Studies of manganese complexes by NMR relaxation rates of water protons.

The interactions of substrates with succinyl-CoA synthetase were investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes. The binding of Mn(II) to the enzyme was investigated by EPR. The effects of phosphorylating the enzyme on its interactions with Mn(II) and substrates were also examined. Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase (E) at approximately four sites with a KD value of 0.14 mM, and the PRR enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8. The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four sites with a KD value of 0.74 mM, and only a small change in epsilonb to 18.1. Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values of epsilont for the ternary E-Mn-ADP complex is 17.0. Free ADP binds about 126 times more weakly to the enzyme than does Mn-ADP. PRR titrations indicated that the values of epsilont for the ternary E-Mn-ADP and (E-P)-Mn-ADP complexes are about the same. Mn-ATP binds very weakly or not at all to (E-P)-Mn.Formation of the ternary complexes of CoA with E-Mn or (E-P)-Mn could be followed by small but significant increases in the PRR enhancement. No ternary complex with succinate could be detected since the addition of succinate had no effect on the PRR enhancement. However, a large decrease in enhancement, at least 2-fold, was observed upon addition of both succinate and CoA. An increase in the PRR enhancement was produced by the interaction of succinyl-CoA with the E-Mn complex. Upper limits of the dissociation constants for CoA from the quaternary E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively. The epsilon values for the quaternary and quinary complexes are 6.4 and 3.1, respectively. The successive occupation of substrate binding sites of succinyl-CoA synthetase produces alterations in the molecular dynamics or in the conformation of the active site (or both), which are accompanied by progressive decreases in the values of epsilon. Thus, the physical parameter used in these studies relects the previously observed catalytic properties of the enzyme system inasmuch as the catalytic function of succinyl-CoA synthetase is potentiated by substrate binding, and catalytic avtivity in partial reactions is maximized as binding sites are successively occupied.     

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)     

Magnetic resonance studies of the manganese guanosine di- and triphosphate complexes with elongation factor Tu.

Analysis of titration data of EF-Tu-GDP with Mn(II) where free and bound Mn(II) were determined by proton relaxation rate of water (PRR) yields one tight Mn(II) binding site and a value of 2 muM for the dissociation constant of Mn(II) from the EF-Tu-MnGDP complex, K'A. The dissociation constant of manganese nucleotide from the ternary EF-Tu-MnGDP complex, K2, 0.2 muM, was derived from the known value of Ks, the dissociation constant for the binary EF-Tu-GDP complex, and the titration data of the ternary complex with excess GDP as titrant. The apparent number, n, of rapidly exchanging water ligands coordinated to bound Mn(II) in the ternary complex EF-Tu-MnGDP is estimated from the frequency dependence of the PRR of the complex to be approximately 1. The value of n and the values of PRR enhancements, epsilont = 4.3 for EF-Tu-MnGDP at 21 degrees, 24.3 MHZ and epsilont = 4.1 for the ternary GTP complex, are unusually low for protein-Mn-nucleotide complexes. The antibiotic X5108 which induces GTPase activity in EF-Tu-MgGTP was shown to bind stoichiometrically to EF-Tu-MnGDP and thereby change the PRR enhancement of the complex from 4.3 to 7.4. The characteristic broad lines in the EPR spectra of Mn(II) nucleotides are strikingly narrowed upon binding of Mn(II) nucleotides to EF-Tu. The long electron spin relaxation times inferred from the EPR spectra indicate a limited access of solvent water to the first coordination sphere of Mn(II) in its EF-Tu-nucleotide complexes. The frequency dependence of the PRR indicates that the electron spin relaxation time, T1e, is the dominant process modulating the Mn(II)-H2O interaction of the EF-Tu-MnGDP complex and consequently determines the correlation time. The value of T1e, estimated from the PRR experiments to be 2.5 ns at 21 degrees, is consistent with the lower limit of T1e obtained from the line widths of the EPR spectrum of the complex. Upon binding of a stoichiometric quantity of the antibiotic X5108, the EPR spectrum of EF-Tu-MnGDP is severely broadened indicating greater access of solvent water to the manganese coordination sphere, i.e. an opening of the nucleotide binding site as already suggested by the increased PRR enhancement.     

Metal ion and substrate binding to bovine galactosyltransferase

Bovine milk galactosyltransferase was examined by ESR and NMR proton relaxation measurements to determine the stoichiometry and nature of manganese and UDP-Gal substrate binding. The ESR and NMR data clearly showed the binding of two (Mn(II) per mol of enzyme in the ternary complex (enzyme-manganese-UDP-Gal). The affinity of the enzyme for manganese is much higher in the presence of UDP-Gal than in its absence. A deenhancement was observed in both water and UDP-Gal proton relaxation rates upon ternary complex formation [enzyme-Mn(II)-UDP-Gal] relative to the metal-substrate [Mn(II)-UDP-Gal] binary complex, yet the temperature dependence of the water proton relaxation rate was consistent with fast exchange. A simple model was proposed which accounted for the pronounced deenhancement, involving a slow conformational interconversion of an initially formed, rapidly exchanging conformer of the enzyme-Mn(II)-UDP-Gal complex to a second form which contributes negligibly to the relaxation.     

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.     

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

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

Mandelate racemase from Pseudomonas putida. Magnetic resonance and kinetic studies of the mechanism of catalysis.

The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated. The enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit with a dissociation constant of 8 muM, in agreement with its kinetically determined activator constant. Also, six additional Mn2+ ions were found to bind to the enzyme, much more weakly, with a dissociation constant of 1.5 mM. Binding to the enzyme at the tight site enhances the effect of Mn2+ on the longitudinal relaxation rate (1/T1p) of water protons by a factor of 11.9 at 24.3 MHz. From the frequency dependence of 1/T1p, it was determined that there are similar to 3 water ligands on enzyme-bound Mn2+ which exchange at a rate larger than or equal to 10-7 sec-1. The correlation time for enzyme-bound Mn2+-water interaction is frequency-dependent, indicating it to be dominated by the electron spin relaxation time of Mn2+. Formation of the ternary enzyme-Mn2+-mandelate complex decreases the number of fast exchanging water ligands by similar to 1, but does not affect tau-c, suggesting the displacement or occlusion of a water ligand. The competitive inhibitors D,L-alpha-phenylglycerate and salicylate produce little or no change in the enzyme-Mn2+-H2O interaction, but ternary complexes are detected indirectly by changes in the dissociation constant of the enzyme-Mn2+ complex and by mutual competition experiments. In all cases the dissociation constants of substrates and competitive inhibitors from ternary complexes determined by magnetic resonance titrations agree with K-M and K-i values determined kinetically and therefore reflect kinetically active complexes. From the paramagnetic effects of Mn2+ on 1/T1 and 1/T2 of the 13C-enriched carbons of 1-[13C]-D,L-mandelate and 2-[13C]-D,L-mandelate, Mn2+ to carboxylate carbon and Mn2+ to carbinol carbon distances of 2.93 plus or minus 0.04 and 2.71 plus or minus 0.04 A, respectively, were calculated, indicating bidentate chelation in the binary Mn2+-mandelate complex. In the active ternary complex of enzyme, Mn2+, and D,L-mandelate, these distances increase to 5.5 plus or minus 0.2 and 7.2 plus or minus 0.2 A, respectively, indicating the presence of at least 98.9% of a second sphere complex in which Mn2+, and C1 and C2 carbon atoms are in a linear array. The water relaxation data suggest that a water ligand is immobilized between the enzyme-bound Mn2+ and the carboxylate of the bound substrate. This intervening water ligand may polarize or protonate the carboxyl group. From 1/T2p the rate of dissociation of the substrate from this ternary complex (larger than or equal to 5.6 times 10-4 sec-1) is at least 52 times greater than the maximal turnover number of the enzyme (1070 sec-1), indicating that the complex detected by nuclear magnetic resonance (NMR) is kinetically competent to participate in catalysis. Relationships among the microscopic rate constants are considered.     

Effects of metals and nucleotides on the inactivation of sea urchin sperm guanylate cyclase by heat and N-ethylmaleimide.

Preincubation of sea urchin sperm guanylate cyclase at 35, 37, 40, or 43 degrees resultedin inactivation. Various metals were able to protect guanylate cyclase against heat inactivation. Estimated binary enzyme-metal dissociation constants for Mn2+, Fe2+, La3+, Ca2+, Ba2+, Mg2+, Co2+, and Ni2+ were 123, 361, 5.5, 692, 984, 335, 79, and 47 muM, respectively. Extrapolated rates of enzyme denaturation in the presence of saturating concentrations of metal divided by the rates of enzyme denaturation in the absence of metal gave values of 0.13, 0.08, minus 0.1, 0.30, 0.59, 0.66, 0.28, and 0.42 for Mn2+, Fe2+, La3+, Ca2+, Ba2+, Mg2+, Co2+, and Ni2+, respectively. GTP, MgGTP, and SrGTP protected the enzyme only slightly against heat inactivation, but CaGTP and MnGTP protected substantially. Neither CaGTP nor MnGTP protected maximally, however, unless the metal concentration exceeded that of GTP. At fixed free Mn2+ or free Ca2+ concentrations, protection curves as a function of MnGTP or CaGTP appeared to be sigmoidal, suggesting multiple nucleotide binding sites. MnATP also protected against heat, but CaATP was virtually ineffective. Sea urchin sperm guanylate cyclase was inactivated by N-ethylmaleimide; CaGTP and MnATP were effective protectants with estimated binary enzyme-Me2+ nucleoside triphosphate dissociation constants of 40 and 170 muM, respectively. MnGTP protected only slightly or not at all against N-ethylmaleimide. These results suggest that: (a) sea urchin sperm guanylate cyclase binds free metal, (b) the binding of free metal is required for protection by nucleotides, and (c) the enzyme contains multiple nucleotide binding sites.     

Nuclear magnetic resonance studies of formyltetrahydrofolate synthetase interactions with formate and methylammonium ion

Using nuclear magnetic resonance techniques, we have measured the internuclear distances separating the nucleotide-bound metal from the carbon and hydrogen nuclei of formate as well as the carbon of methylammonium cation when bound to formyltetrahydrofolate synthetase. Measurements were made of the paramagnetic effect on the spin-lattice relaxation rates (1/T1) of 13C and 1H nuclei arising from the replacement of Mg2+ with Mn2+, which binds to the enzyme in the form of a metal-nucleotide complex. Distances from Mn2+ to the formate carbon and proton were found to be 6.3 and 7.4 A, respectively, in the E . ATP . Mn2+ . formate complex and 6.0 and 7.1 A, respectively, in the E . ADP . Mn2+ . formate complex. When tetrahydrofolate was added to the latter complex, the exchange of formate was greatly reduced and became rate limiting for relaxation. These results are consistent with substantial conformational effects produced by the binding of the cofactor. The distance from Mn2+ to the methylammonium carbon in the E . ADP . Mn2+ . CH3NH+3, E . ADP . Mn2+ . formate . CH3NH3+, and E . ADP . Mn2+ . tetrahydrofolate . CH3NH3+ complexes was estimated to be in the range of 7.4-12 A. However, in the E . ADP . Mn2+ formate . tetrahydrofolate . CH3NH3+ complex, the data suggest that exchange of cation contributes significantly to relaxation. These results, combined with other known features of the enzyme, suggest that there may be a monovalent cation site within the active site of the enzyme.     

Investigations of equilibrium complexes of myoxin subfragment 1 with the manganous ion and adenosine diphosphate using magnetic resonance techniques.

Electron paramagnetic resonance spectroscopy and water proton relaxation rate (PRR) measurements were used to characterize a complex formed at the myosin subfragment 1 (S1) ATPase site with stoichiometric amounts of Mn(II) and ADP. In the absence of nucleotide, Mn(II) binding at the active site is very weak, although two other classes of sites for Mn(II) on subfragment 1 were identified which are not directly involved in the ATPase reaction. A high affinity Mn(II) site (termed L-site with KL = 3 muM) is associated with a region of the molecule which is susceptible to proteolysis (probably the LC2 light chain subunit) since its stoichiometry depends on the conditions employed for the preparation of subfragment 1 during the papain treatment of myosin. In addition there are a number of weak sites for Mn(II) (termed N-sites) probably associated with anionic groups on the surface of the molecule. In order to study the properties of Mn(II) and ADP bound at the active site by magnetic resonance techniques, subfragment 1 preparations virtually free of the L-site were used, since such an ancillary site competes for the available Mn(II). MnADP binds to subfragment 1 with an apparent dissociation constant, KT, of about 4 muM at 25 degrees. The resultant complex, S1-MnADP, has a low PRR enhancement factor (1.7 at 24.3 MHZ), and its frequency (magnetic field) dependence indicates that this is because there are no readily exchangeable water molecules within the first coordination sphere of Mn(II. Relaxation of the bulk solvent is mediated by protons bound transiently within the outer spheres (4 to 7 A) of the Mn(II). A nitroxide spin label attached to the reactive thiol group of subfragment 1 enhances the solvent PRR, and this property is sensitive to the binding of MgADP to the active site. However, no dipolar spin-spin interaction was detected between the nitroxide group and Mn(II) in the S1-MnADP complex, indicating that the metal ion and thiol group are well separated.     

Role of divalent metals in the kinetic mechanism of insulin receptor tyrosine kinase

The kinetic and thermodynamic interrelationships of peptide substrate (Val5-angiotensin 11), metal-ATP, and divalent metal cations with rat liver insulin receptor tyrosine kinase (IRTK) were investigated. Results of the initial rate studies with varying peptide and MnATP substrates indicates that the kinetic mechanism for IRTK is of the sequential type and therefore rules out a ping pong Bi Bi pathway. Hence, peptide substrate and metal-ATP bind to the kinase prior to the release of products. MnADP was a linear competitive inhibitor of MnATP and a noncompetitive inhibitor of peptide substrate. A synthetic tyrosine-containing pentapeptide, Glu-Glu-Phe-Tyr-Phe (EEFYF), was a linear competitive inhibitor of peptide substrate and a noncompetitive inhibitor of MnATP. Accordingly, the data show that phosphorylation of peptide substrate occurs via a rapid random equilibrium Bi Bi mechanism in which the kinase has the potential to react initially with either of the two substrates. In contrast, divalent metal cations and metal-ATP were found to interact with the kinase in a mutually inclusive manner, with metal binding to the kinase prior to MnATP. It was also found that divalent metals increase the affinity of the kinase for metal-ATP but do not affect the affinity of IRTK for metal-ADP product. Hence, divalent metals, during the reaction of association of enzyme with one of its substrates to form the binary complex, increase the relative concentration of E-ATP complex versus E-peptide complex, thus introducing a thermodynamic-dependent ordering for the interaction of substrates with the enzyme. To investigate the thermodynamics of this system, we assumed that under initial conditions the kinetic data we obtained reflected the association constants of reactants with the enzyme.     

Proton magnetic relaxation studies of the interaction of D-xylose and xylitol with D-xylose isomerase. Characterization of metal-enzyme-substrate interactions.

The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the competitive inhibitor xylitol has been examined by nuclear magnetic resonance. A greater paramagnetic effect of enzyme-bound Mn2+ on the alpha anomer of D-xylose than on the beta anomer was observed, providing independent evidence for the specificity of D-xylose isomerase for the alpha anomeric form of D-xylose. The exchange rate of alpha-D-xylose into the ternary complex, determined from the normalized paramagnetic contribution to the transverse relaxation rate (1/fT2p) of the carbon 1 proton of alpha-D-xylose, exceeds Vmax for the enzymatic reaction by 3 orders of magnitude. The amount of xylitol necessary to displace alpha-D-xylose from the substrate-enzyme-Mn2+ complex is consistent with the Km value for alpha-D-xylose and the inhibitor constant Ki for xylitol previously determined by the methods of enzyme kinetics. These results suggest that the NMR experiments observe complexes of D-xylose isomerase which are kinetically and thermodynamically competent to participate in catalysis. From the frequency dependence of the paramagnetic contribution to the longitudinal relaxation rate (1/T1p) of the carbon 1 proton of alpha-D-xylose, the correlation time (tauc) which modulates the dipolar interaction between enzyme-bound Mn2+ and alpha-D-xylose has been determined (5.1 x 1o(-10) s). From these observations a range of calculated distances between enzyme-bound Mn2+ and the carbon 1 proton of alpha-D-xylose (9.1 +/- 0.7 A) has been found. The enzyme-bound Mn2+ has comparable effects on the carbon 1, carbon 2, and carbon 5 protons of alpha-D-xylose, suggesting that these protons of the enzyme-bound substrate are equidistant from the bound Mn2+. A similar distance (9.4 +/- 0.7 A) between the enzyme-bound Mn2+ and the terminal methylene protons of xylitol, an analog of the open chain intermediate in the reaction, has been determined. The results of the present substrate relaxation and previous water relaxation studies suggest that two small ligands such as water molecules or a large portion of the protein intervene between the bound metal ion and the bound substrate in the active ternary complex.     

Magnetic resonance study of the three-dimensional structure of creatine kinase-substrate complexes. Implications for substrate specificity and catalytic mechanism.

The paramagnetic effects of the bound manganese ion and of a covalently attached spin label on proton nuclear spin relaxation rates have been used to calculate distances for a structural model of the MnADP and creatine complexed to creatine kinase from rabbit muscle. The nucleotide and guanidino substrates are so aligned on the enzyme that the transferable phosphoryl group on one substrate is in apposition to the acceptor moiety on the second substrate. The divalent metal ion is most probably liganded to the alpha and beta phosphates of the nucleotide substrate, both in the abortive MnADP-creatine-enzyme complex and in the active MnATP-creatine-enzyme complex. The metal ion-formate distance approximately 5 A in the Mn(II)ADP-formate-creatine-enzyme complex and less than 5 A in the Co(II)ADP-formate-creatine-enzyme complex is consistent with the suggestion that the monovalent anion is binding at the site normally occupied by the transferable phosphoryl group, thus producing a complex which mimics the transition state. Although only an upper limit of the distance from Mn(II) to the guanidino substrate could be determined in the presence of formate, it could be concluded that the disposition of the guanidino substrate changes upon addition of formate, since the relative distances of the methyl and methylene group are inverted. The effect of formate and nitrate on increasing the residence time of creatine in the MnADP-creatine-enzyme complex as determined by NMR provides evidence that the complexes observed by NMR are identical with those involved in the catalytic mechanism, since a parallel effect of formate and nitrate is observed in the kinetics of the enzymatic reaction, where the dissociation constant of creatine from the abortive quaternary complex decreases in the presence of the anions as had been determined from their inhibition of the forward reaction (Milner-White, E.J., and Watts, D.C. (1971) Biochem. J. 122, 727-740). Although the guanidino substrate is not directly liganded to the divalent metal ion, the electron paramagnetic resonance spectrum of manganese in the transition state analog complexes, i.e. nitrate-ADP-guanidino substrate-enzyme, is strongly dependent on catalytic activity of the guanidino substrate. The structural differences observed by EPR among transition state analog complexes with various guanidino substrates were not reflected in distances from Mn(II) to the guanidino substrate, which were 10% and 0.3% as active as creatine. Within the experimental error of 1 A, the distances were the same. The enzyme or the enzyme-substrate complexes may be considered to exist in a number of structurally distinct conformations in equilibrium based on the EPR spectra and on the anomalous temperature-dependence of the relaxation rates of the formate proton of the transition state analog complexes...     

Human leukemic cells resistant to 5-fluoro-2'-deoxyuridine contain a thymidylate synthetase with lower affinity for nucleotides

A line of human lymphocytic leukemia cells (CCRF-CEM) has been obtained which is 140-fold resistant to the potent cell growth inhibitor 5-fluoro-2'-deoxyuridine (FdUrd). The cells were also 11-fold cross-resistant to 5-fluorouracil. In contrast to several previous studies involving FdUrd-resistant mouse cells, thymidylate synthetase levels were not substantially elevated in these FdUrd-resistant human leukemic cells. Thymidine kinase activity was also unchanged in the resistant cells, although the levels of 5-fluoro-2'-deoxyuridylate (FdUMP), the potent inhibitor of thymidylate synthetase, generated at equimolar doses of FdUrd were about 40% lower than in the sensitive cells. Studies of the kinetics of FdUMP binding to thymidylate synthetase isolated from the FdUrd-resistant cells disclosed a considerably higher dissociation constant (Kd = 1.0 X 10(-9) M) for the ternary covalent enzyme . FdUMP . 5,10-methylene tetrahydrofolate complex compared to the value obtained with enzyme from sensitive cells (Kd = 4.4 X 10(-11) M). The thymidylate synthetase from the FdUrd-resistant cells also showed 17-fold weaker binding of 2'-deoxyuridylate, even though the Km value for 2'-deoxyuridylate was 3-fold lower compared to the enzyme from FdUrd-sensitive cells. The turnover number of the altered enzyme was 1.8-fold higher than that for the normal enzyme but the rate constants for the release of FdUMP from the ternary complex, which is also an enzyme-catalyzed reaction, were identical for both enzymes. Electrophoresis of the radiolabeled ternary complexes on nondenaturing gels showed small but reproducible differences in migration rates. These results demonstrate that the mechanism of resistance to FdUrd in this cell line involves an alteration in the target enzyme, thymidylate synthetase, which causes it have a lower affinity for nucleotides.     

Kinetic and magnetic resonance studies of effects of genetic substitution of a Ca2+-liganding amino acid in staphylococcal nuclease

The X-ray structure of staphylococcal nuclease suggests octahedral coordination of the essential Ca2+, with Asp-21, Asp-40, and Thr-41 of the enzyme providing three of the six ligands [Cotton, F. A., Hazen, E. E., Jr., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555]. The Asp-40 codon was mutated to Gly-40 on the gene that had been cloned into Escherichia coli, and the mutant (D40G) and wild-type enzymes were both purified from E. coli by a simple procedure. The D40G mutant forms a (5 +/- 2)-fold weaker binary complex with Ca2+ as found by kinetic analysis and by Ca2+ binding studies in competition with Mn2+, a linear competitive inhibitor. Similarly, as found by electron paramagnetic resonance (EPR), Mn2+ binds to the D40G mutant with a 3-fold greater KD than that found with the wild-type enzyme. These differences in KD are increased by saturation of staphylococcal nuclease with the DNA substrate such that KmCa is 10-fold greater and KIMn is 15-fold greater for the mutant than for the wild-type enzyme, although KMDNA is only 1.5-fold greater in the mutant. The six dissociation constants of the ternary enzyme-Mn2+-nucleotide complexes of 3',5'-pdTp and 5'-TMP were determined by EPR and by paramagnetic effects on 1/T1 of water protons, and the dissociation constants of the corresponding Ca2+ complexes were determined by competition with Mn2+. Only small differences between the mutant and wild-type enzymes are noted in K3, the dissociation constant of the nucleotides from their respective ternary complexes. 3',5'-pdTp raises the affinities of both wild-type and mutant enzymes for Mn2+ by factors of 47 and 31, respectively, while 5'-TMP raises the affinities of the enzymes for Mn2+ by smaller factors of 6.8 and 4.4, respectively. Conversely, Mn2+ raises the affinities of both wild-type and mutant enzymes for the nucleotides by 1-2 orders of magnitude. Analogous effects are observed in the ternary Ca2+ complexes. Dissociation constants of Ca2+ and Mn2+ from binary and ternary complexes, measured by direct binding studies, show reasonable agreement with those obtained by kinetic analysis. Structural differences in the ternary metal complexes of the D40G mutant are revealed by a 31-fold decrease in Vmax with Ca2+ and by 1.4-3.1-fold decreases in the enhancement of 1/T1 of water protons with Mn2+.(ABSTRACT TRUNCATED AT 400 WORDS)