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.     

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.     

Kinetic and magnetic resonance studies of the role of metal ions in the mechanism of Escherichia coli GDP-mannose mannosyl hydrolase, an unusual nudix enzyme

Escherichia coli GDP-mannose mannosyl hydrolase (GDPMH), a homodimer, catalyzes the hydrolysis of GDP-alpha-D-sugars to yield the beta-D-sugar and GDP by nucleophilic substitution with inversion at the C1' carbon of the sugar [Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Biochemistry 39, 8603-8608]. GDPMH requires a divalent cation for activity such as Mn2+ or Mg2+, which yield similar kcat values of 0.15 and 0.13 s(-1), respectively, at 22 degrees C and pH 7.5. Kinetic analysis of the Mn2+-activated enzyme yielded a K(m) of free Mn2+ of 3.9 +/- 1.3 mM when extrapolated to zero substrate concentration (K(a)Mn2+), which tightened to 0.32 +/- 0.18 mM when extrapolated to infinite substrate concentration (K(m)Mn2+). Similarly, the K(m) of the substrate extrapolated to zero Mn2+ concentration (K(S)(GDPmann) = 1.9 +/- 0.5 mM) and to infinite Mn2+ concentration (K(m)(GDPmann) = 0.16 +/- 0.09 mM) showed an order of magnitude decrease at saturating Mn2+. Such mutual tightening of metal and substrate binding suggests the formation of an enzyme-metal-substrate bridge complex. Direct Mn2+ binding studies, monitoring the concentration of free Mn2+ by EPR and of bound Mn2+ by its enhanced paramagnetic effect on the longitudinal relaxation rate of water protons (PRR), detected three Mn2+ binding sites per enzyme monomer with an average dissociation constant (K(D)) of 3.2 +/- 1.0 mM, in agreement with the kinetically determined K(a)Mn2+. The enhancement factor (epsilon(b)) of 11.5 +/- 1.2 indicates solvent access to the enzyme-bound Mn2+ ions. No cross relaxation was detected among the three bound Mn2+ ions, suggesting them to be separated by at least 10 A. Such studies also yielded a weak dissociation constant for the binary Mn2+-GDP-mannose complex (K1 = 6.5 +/- 1.0 mM) which significantly exceeded the kinetically determined K(m) values of Mn2+, indicating the true substrate to be GDP-mannose rather than its Mn2+ complex. Substrate binding monitored by changes in 1H-15N HSQC spectra yielded a dissociation constant for the binary E-GDP-mannose complex (K(S)(GDPmann)) of 4.0 +/- 0.5 mM, comparable to the kinetically determined K(S) value (1.9 +/- 0.5 mM). To clarify the metal stoichiometry at the active site, product inhibition by GDP, a potent competitive inhibitor (K(I) = 46 +/- 27 microM), was studied. Binding studies revealed a weak, binary E-GDP complex (K(D)(GDP) = 9.4 +/- 3.2 mM) which tightened approximately 500-fold in the presence of Mn2+ to yield a ternary E-Mn2+-GDP complex with a dissociation constant, K3(GDP) = 18 +/- 9 microM, which overlaps with the K(I)(GDP). The tight binding of Mn2+ to 0.7 +/- 0.2 site per enzyme subunit in the ternary E-Mn2+-GDP complex (K(A)' = 15 microM) and the tight binding of GDP to 0.8 +/- 0.1 site per enzyme subunit in the ternary E-Mg2+-GDP complex (K3 < 0.5 mM) indicate a stoichiometry close to 1:1:1 at the active site. The decrease in the enhancement factor of the ternary E-Mn2+-GDP complex (epsilon(T) = 4.9 +/- 0.4) indicates decreased solvent access to the active site Mn2+, consistent with an E-Mn2+-GDP bridge complex. Fermi contact splitting (4.3 +/- 0.2 MHz) of the phosphorus signal in the ESEEM spectrum established the formation of an inner sphere E-Mn2+-GDP complex. The number of water molecules coordinated to Mn2+ in this ternary complex was determined by ESEEM studies in D2O to be two fewer than on the average Mn2+ in the binary E-Mn2+ complexes, consistent with bidentate coordination of enzyme-bound Mn2+ by GDP. Kinetic, metal binding, and GDP binding studies with Mg2+ yielded dissociation constants similar to those found with Mn2+. Hence, GDPMH requires one divalent cation per active site to promote catalysis by facilitating the departure of the GDP leaving group, unlike its homologues the MutT pyrophosphohydrolase, which requires two, or Ap4A pyrophosphatase, which requires three.     

Characterization of ATP binding sites of sheep kidney medulla (Na+ + K+)--ATPase using CrATP

The nucleotide substrate sites of sheep kidney medulla (NA+ + K+)-ATPase are characterized using CrATP, a paramagnetic, substitution-inert substrate analogue probe. The paramagnetic effect of CrATP on 1/T1 of water protons of water protons is enhanced upon complexation with the enzyme. Titrations of the enzyme with CrATP in the presence of Na+ and K+ yielded characteristic enhancements for the binary enzyme-CrATP and ternary enzyme-Mg2+-CrATP complexes of 3.3 and 3.6 and dissociation constants for CrATP of 5 and 12 microM, respectively. Substitution of Li+ for K+ in these titrations did not substantially alter the titration behavior. From the frequency dependence of 1/T1, the correlation time, tau c, for the dipolar water proton-CrATP interaction is 2.7 x 10(-10) sec, indicating that tau c is dominated by tau s, the electron spin relaxation time of Cr3+. The paramagnetic effect of enzyme-bound Mn2+ on 1/T1 of water protons decreases upon the addition of CrATP. Titration of the binary enzyme-Mn2+ complex with CrATP decreases the characteristic enhancement due to Mn2+ from 6.6-8.0 to 1.5. The failure to observe free Mn2+ epr signals in solutions of the ATPase, Mn2+, and CrATP demonstrate that this decrease in epsilon Mn is due to cross-relaxation between Mn2+ and Cr3+ bound simultaneously to the enzyme, and not to displacement of Mn2+ from the enzyme by CrATP. The relaxation rate, 1/T1, of 7Li+ is increased upon addition of CrATP to solutions of the ATPase, indicating that the sites for Li+ and CrATP are close on the enzyme. A Cr3+-Li+ distance of 4.8 +/- 0.5 angstrom is calculated from that data.     

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.     

Phosphorus-31 nuclear relaxation rate studies of the nucleotides on phosphoenolpyruvate carboxykinase

The interactions of nucleotide substrates with the enzyme phosphoenolpyruvate carboxykinase and its Mn2+ complex were investigated by several methods. Direct binding shows the formation of stoichiometric complexes. The presence of Mn2+ increases the affinity of the enzyme for nucleotide. A higher affinity for GTP (Kd less than 2 microM) than for GDP (Kd = 15 microM) was determined. Solvent proton relaxation rate studies indicate no substantial difference in titration curves for free nucleotide or for Mg-nucleotide to the enzyme-Mn complex. The effect of Mn2+ on the 31P relaxation rates of IDP and of ITP in the binary Mn-nucleotide complex indicates the formation of direct coordination complexes. The distances of the alpha- and beta-31P of IDP to Mn2+ are identical (3.5 A). The Mn2+ distance to the beta- and gamma-31P of ITP is also identical (3.7 A) and is 0.2 A further from the alpha-phosphorus. In the presence of P-enolpyruvate carboxykinase, the effect of Mn2+ on the 31P relaxation rates was measured at 40.5 MHz and at 121.5 MHz. The dipolar correlation time was calculated to be 0.6-5.4 ns, depending upon assumptions made. The Mn2+ to phosphorus distances indicate the nucleotide substrates form a second sphere complex to the bound Mn2+. From 1/T2 measurements, electron delocalization from Mn2+ to the phosphorus atoms is indicated; this effect occurs although direct coordination does not take place. The exchange rate of GTP from the enzyme-Mn complex (koff = 4 X 10(4) s-1) is rapid compared to kcat with a lower energy of activation (9.2 kcal/mol) than for catalytic turnover.(ABSTRACT TRUNCATED AT 250 WORDS)     

The divalent cation dependence of bovine brain calmodulin-dependent phosphatase

The divalent cation dependence of a calmodulin-stimulated phosphatase from bovine brain has been characterized kinetically using phosphorylated myelin basic protein and casein as substrates. At saturating concentrations of calmodulin, dephosphorylation of both myelin basic protein and casein was catalyzed 8- to 10-fold more rapidly at saturating concentrations of Mn2+ than at saturating concentrations of Ca2+. Half-maximal rates of dephosphorylation of both substrates occurred at either 15 microM Mn2+ or 1 microM Ca2+, and the Kact for each ion was not influenced appreciably by the presence of calmodulin. Half-maximal rates of dephosphorylation were observed at concentrations of calmodulin ranging from 3 X 10(-8) to 10(-6) M at saturating concentrations of divalent cations depending on the substrate used and the particular cation chosen. Trypsin treatment of the phosphatase activated the enzyme several-fold, eliminated its calmodulin dependence, but did not alter the Mn2+ concentration dependence of the activity. Ca2+ (10 microM) increased dephosphorylation rates without altering the Mn2+ concentration dependence of the phosphatase activity regardless of the presence of calmodulin. Mg2+ at millimolar concentrations did not alter the Ca2+ or Mn2+ concentration dependence of the activity. As measured without calmodulin, Ca2+ (90 microM) or Mn2+ (200 microM) produced nearly identical alterations of the far ultraviolet circular dichroic spectrum of the phosphatase.     

Metal-ion-promoted binding of triazine dyes to proteins. The interaction of Cibacron Blue F3G-A with yeast hexokinase.

Bivalent metal ions, particularly Zn2+ and other members of the first-row transition series, promote irreversible inactivation of yeast hexokinase by Cibacron Blue F3G-A at a site competitive with both ATP and D-glucose. Difference spectroscopy indicates that the protein-dye dissociation constant is decreased from 250 micrometers in the absence of metal ions to less than 100 micrometers in the presence of appropriate concentrations of metal ions, with specificity displayed in the sequence of Zn2+ greater than Cu2+ greater than Ni2+ greater than Mn2+. Quantitative inactivation of yeast hexokinase leads to the incorporation of approx. 1 mol of Cibacron Blue F3G-A/mol of subunit of mol. wt. 51 000 in both the presence and the absence of metal ion. These results suggest the formation of a highly specific ternary complex involving enzyme, dye and metal ion at the active-site region of the enzyme, and correlate well with the known effects of metal ions in promoting the binding of hexokinase to immobilized Cibacron Blue F3G-A.     

Conformational changes required for pyruvate kinase activity as modulated by monovalent cations.

The interaction of a series of alkylamines with muscle pyruvate kinase was investigated by kinetic and physical studies in order to understand the mechanisms by which certain monovalent cations can activate the enzyme and to define several of the important conformational changes necessary for catalytic activity. Monomethylammonium ion interacts with pyruvate kinase to activate the enzyme. Dimethyland trimethylammonium ions do not activate, but are competitive inhibitors against activating cations. Tetramethylammonium ion neither activates nor inhibits pyruvate kinase activity. When the enzyme is in the presence of monomethylammonium ion or dimethylammonium ion, a conformational change is observed by ultraviolet difference spectroscopy. This conformational change is similar to that observed with other activating cations and appears to be a necessary but no sufficient conformational change in the formation of an active complex. The interaction of the substrate phosphoenolpyruvate with the pyruvate kinase-Mn2+ complex in the presence of these cations was studied by water proton relaxation rate measurements. The affinity of the enzyme-Mn2+ complex for phosphoenolpyruvate is decreased by a factor of 5 in the presence of any of the alkylamines compared to the affinity measured in the presence of K+ or NH4+. No change in the Km of phosphoenolpyruvate is observed however when it is measured in the presence of monomethylammonium ion, suggesting that the decrease in affinity for the substrate is not the reason for lack of enzymic activity. The conformation of the ternary enzyme-Mn2+-phosphoenolpyruvate complex about the bound Mn2+, as reflected by the enhancement values (epsilont) measured, differs depending upon the nature of the monovalent cation. The epsilon t values measured in the presence of the alkylamines are larger (epsilont - 5.7 +/- 0.2) than those measured in the presence of K+ or NH4+ (epsilont = 1.9 +/- 0.1).     

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.     

Biochemical characterization of cytosolic fructose-1,6-bisphosphatase from apple (Malus domestica) leaves

Cytosolic fructose-1,6-bisphosphatase was purified to apparent homogeneity from the leaves of apple, a sorbitol synthesizing species. The enzyme was a homotetramer with a subunit mass of 37 kDa, and was highly specific for fructose 1,6-bisphosphate (F1,6BP) with a Km of 3.1 micro M and a Vmax of 48 units (mg protein)(-1). Either Mg2+ or Mn2+ was required for its activity with a Km of 0.59 mM and 62 micro M, respectively. Li+, Ca2+, Zn2+, Cu2+ and Hg2+ inhibited whereas Mn2+ enhanced the Mg2+ activated enzyme activity. Fructose 6-phosphate (F6P) was found to be a mixed type inhibitor with a Ki of 0.47 mM. Fructose 2,6-bisphosphate (F2,6BP) competitively inhibited the enzyme activity and changed the substrate saturation curve from hyperbolic to sigmoidal. AMP was a non-competitive inhibitor for the enzyme. F6P interacted with F2,6BP and AMP in a synergistic way to inhibit the enzyme activity. Dihydroxyacetone phosphate slightly inhibited the enzyme activity in the presence or absence of F2,6BP. Sorbitol increased the susceptibility of the enzyme to the inhibition by high concentrations of F1,6BP. High concentrations of sorbitol in the reaction mixture led to a reduction in the enzyme activity.     

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.     

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

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

Stimulation of calcium transport of sarcoplasmic reticulum vesicles by the calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N',-tetraacetic acid

The calcium ion dependence of calcium transport by isolated sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been investigated by means of the Calcium-stat method, in which transport may be measured in the micromolar free calcium ion concentration range, in the absence of calcium buffers. At pH 7.2 and 20 degrees C, ATP, in the range 1 to 10 mM, decreased [Ca2+]0.5 from 2.0 microM to 0.3 microM and decreased Vmax of oxalate-supported transport from 0.5 to 1.3 mumol min-1 mg-1. Simultaneous measurements of transport and of ATPase activity in the range 0.8 to 10 microM free Ca2+ showed a ratio of 2.1 calcium ions translocated/molecule of ATP hydrolyzed. Transport, in the presence of 5 mM ATP, ceased when calcium ion concentration fell to 0.6 to 1.2 microM, whilst ATPase activity of 90 nmol of ATP hydrolyzed min-1 mg-1 persisted. The data obtained by the Calcium-stat method differed from those described previously using calcium buffers, in that they showed lower apparent affinities of the transport site for calcium ions, more marked sigmoidal behavior, an effect of ATP concentration on Ca2+ concentration dependence and lower ATPase activity in the absence of transport. The calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (CaEGTA) had no effect when transport was stimulated maximally at saturating free Ca2+ concentrations. However, at calcium ion levels below [Ca2+]0.5, 70 microM CaEGTA stimulated transport to rates of 20 to 45% of Vmax. Half-maximal stimulation of transport occurred at 19 microM CaEGTA. CaEGTA, 50 microM, decreased [Ca2+]0.5, determined at 5 mM ATP, from 1.3 microM to 0.45 microM. It is proposed that a ternary complex, E . Ca2+ . EGTA4-, is formed as an intermediate species during CaEGTA-stimulated calcium transport by sarcoplasmic reticulum membranes and stimulates the calcium pump at limiting free Ca2+ ion concentration.     

Nuclear magnetic resonance studies of fructose 2,6-bisphosphate and adenosine 5'-monophosphate interaction with bovine liver fructose-1,6-biphosphatase

1H and 31P nuclear magnetic resonance was used to investigate the interaction of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) with bovine liver fructose-1,6-bisphosphatase. Mn2+ bound to fructose-1,6-bisphosphatase was used as a paramagnetic probe to map the active and AMP allosteric sites of fructose-1,6-bisphosphatase. Distances between enzyme-bound Mn2+ and the phosphorus atoms at C-6 of fructose-6-P and alpha-methyl-D-fructofuranoside 1,6-bisphosphate were identical, and the enzyme-Mn to phosphorus distance determined for the C-6 phosphorus atom of Fru-2,6-P2 was very similar to these values. Likewise, the enzyme-Mn to phosphorus distances for Pi, the C-1 phosphorus atom of alpha-methyl-D-fructofuranoside 1,6-bisphosphate, and the C-2 phosphorus atom of Fru-2,6-P2 agreed within 0.5 A. The distance between enzyme-bound Mn2+ and the phosphorus atom of AMP was significantly shorter than the distances obtained for any of the aforementioned ligands, but the presence of Fru-2,6-P2 caused the enzyme-Mn to phosphorus distance for AMP to lengthen markedly. NMR line broadening of AMP protons was studied at various temperatures. The dissociation rate constant was found to be greater than 20 s-1. It was concluded that Fru-2,6-P2 strongly affects the interaction of AMP with fructose-1,6-bisphosphatase and that the sugar most likely acts at the active site of the enzyme.     

Magnetic resonance and kinetic studies of the partial complex and Component I subunit forms of Salmonella typhimurium anthranilate synthase

Metal ion interactions of the monofunctional partial complex of Salmonella typhimurium anthranilate synthase were investigated using kinetic, NMR, and EPR methods. Mn2+ activates AS-partial complex in place of Mg2+, with a Km of 0.08 microM for Mn2+ and of 3.5 microM for Mg2+ in glutamine-dependent anthranilate synthase activity. The kinetics indicated that the metal interacts at the active site with chorismate, not glutamine. EPR and NMR water proton relaxation rate (PRR) studies supported this conclusion. EPR binding analysis showed that chorismate dramatically tightens Mn2+ binding by the partial complex. PRR experiments indicated that stoichiometric amounts of chorismate cause a substantial decrease in the enhancement of water relaxation by Mn2+, while millimolar amounts of glutamine have no effect. Analysis of the frequency dependence of water proton relaxation rates yielded dipolar correlation times of 2.5 x 10(-9) s and 4.1 x 10(-9) s for the Mn2+-partial complex and Mn2+-partial complex-chorismate complexes, respectively. These studies also indicated that chorismate binding reduces the number of fast-exchanging water molecules on enzyme-bound Mn2+ from 1 to 0.25. PRR experiments with the native bifunctional anthranilate synthase-phosphoribosyltransferase enzyme indicated the existence of additional Mn2+-binding sites which presumably function to activate the phosphoribosyltransferase activity of the Component II subunit.     

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Characterization of assembly, product formation, and heparin dissociation steps in the factor Xa reaction

The kinetics of alpha-factor Xa inhibition by antithrombin III (AT) were studied in the absence and presence of heparin (H) with high affinity for antithrombin by stopped-flow fluorometry at I 0.3, pH 7.4 and 25 degrees C, using the fluorescence probe p-aminobenzamidine (P) and intrinsic protein fluorescence to monitor the reactions. Active site binding of p-aminobenzamidine to factor Xa was characterized by a 200-fold enhancement and 4-nm blue shift of the probe fluorescence emission spectrum (lambda max 372 nm), 29-nm red shift of the excitation spectrum (lambda max 322 nm), and dissociation constant (KD) of about 80 microM. Under pseudo-first order conditions [( AT]0, [H]0, [P]0 much greater than [Xa]0), the observed factor Xa inactivation rate constant (kobs) measured by p-aminobenzamidine displacement or residual enzymatic activity increased linearly with the "effective" antithrombin concentration (i.e. corrected for probe competition) up to 300 microM in the absence of heparin, indicating a simple bimolecular process with a rate constant of 2.1 x 10(3) M-1 s-1. In the presence of heparin, a similar linear dependence of kobs on effective AT.H complex concentration was found up to 25 microM whether the reaction was followed by probe displacement or the quenching of AT.H complex protein fluorescence due to heparin dissociation, consistent with a bimolecular reaction between AT.H complex and free factor Xa with a 300-fold enhanced rate constant of 7 x 10(5) M-1 s-1. Above 25 microM AT.H complex, an increasing dead time displacement of p-aminobenzamidine and a downward deviation of kobs from the initial linear dependence on AT.H complex concentration were found, reflecting the saturation of an intermediate Xa.AT.H complex with a KD of 200 microM and a limiting rate of Xa-AT product complex formation of 140 s-1. Kinetic studies at catalytic heparin concentrations yielded a kcat/Km for factor Xa at saturating antithrombin of 7 x 10(5) M-1 s-1 in agreement with the bimolecular rate constant obtained in single heparin turnover experiments. These results demonstrate that 1) the accelerating effect of heparin on the AT/Xa reaction is at least partly due to heparin promoting the ordered assembly of antithrombin and factor Xa in an intermediate ternary complex and that 2) heparin catalytic turnover is limited by the rate of conversion of the ternary complex intermediate to the product Xa-AT complex with heparin dissociation occurring either concomitant with this step or in a subsequent faster step.     

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)     

Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.     

A one-to-one Mg2+:Mn2+ exchange in rat erythrocytes

Mg2+ efflux in rat erythrocytes was stimulated by increases in external Na+ concentration following a Michaelian-like function with an apparent dissociation constant (KNa) of 11 +/- 3 mM (mean +/- S.D. of three experiments) and a variable maximal rate ranging from 150 to 1200 mumol (liter (1) cells X h)-1. Na+-stimulated Mg2+ efflux was inhibited by quinidine and by ATP depletion. In the absence of external Na+, Mg2+ efflux was stimulated by increases in external Mn2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMn) of 35 +/- 15 microM (mean +/- S.D. of four experiments) and a variable maximal rate ranging from 350 to 1400 mumol (1 cells X h)-1. Mn2+-stimulated Mg2+ efflux was inhibited by quinidine, by ATP depletion, and by increasing the external Na+ concentration. Quinidine-sensitive (or ATP-dependent) Mg2+ efflux exhibited very similar values when compared with quinidine-sensitive (or ATP-dependent) Mn2+ influx. Mn2+ efflux in rat erythrocytes (loaded with total internal Mn2+ contents of 230-450 mumol/l cells) was stimulated by increases in external Na+ concentration and inhibited by quinidine. In the absence of external Na+, Mn2+ efflux was stimulated by increases in external Mg2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMg) of about 35 +/- 5 microM (mean +/- range of two experiments) and a maximal rate of about 60-100 mumol (1 cells X h)-1. In conclusion, the Na+-stimulated Mg2+ carrier of rat erythrocytes may catalyze a one-to-one and reversible Mn2+:Mg2+ exchange in the absence of external Na+.