George H. Reed

EPR of Mn(II) Complexes with Enzymes and Other Proteins

The history of EPR applications to the study of manganese dates to the very first successful resonance experiments by Zavoisky (1945). Since then there have been numerous EPR studies of Mn(II) in diverse materials. The reader may find references to many of these studies in review articles and monographs on EPR (Abragam and Bleaney, 1970; Goodman and Raynor, 1970; Konig, 1968; Kaiser and Kevan, 1968). Applications of EPR to studies of Mn(II) complexes with proteins began with studies by Malmstrom et al. (1958) wherein the strong isotropic EPR signal for “unbound” Mn(H2O) 6 2+ was used to determine dissociation constants for Mn(II)-protein complexes. Such analytical applications of the EPR signal for Mn(H2O) 6 2+ (Cohn and Townsend, 1954) for measuring dissociation constants have continued. However, during the last decade the EPR signals for the protein-bound Mn(II) have been measured for enzymes and other proteins, and this latter application is the basis for the present chapter.

The significance of the slow dissociation of divalent metal ions from myosin 'regulatory' light chains.

It is well established that Ca2+ binding to troponin C, located on the actin filament, is involved in the activation of contraction of vertebrate skeletal muscle by relieving the inhibitory effect of the troponintropomyosin system [ 11. More recently the discovery of a Ca2+ binding site on the DTNB light chain of the myosin filament has led to the proposal of this being an additional site engaged in the control of contraction [2,3]. These ideas received impetus from the finding that the vertebrate DTNB light chain can substitute for the regulatory light chain of scallop myosin in conferring Ca2+ sensitivity to the ATPase of scallop myofibrils, although the DTNB light chain does not appear to participate directly [4]. Vertebrate skeletal myosin itself does not exhibit a Ca2+ sensitive, actinactivated ATPase in vitro in the absence of troponin and tropomyosin [5,6]. Nevertheless it remains plausible that Ca2+ binding to the DTNB light chain initiates the movement of the myosin crossbridges towards the actin filaments [2,3,7] and such an effect might not be revealed by the actomyosin ATPase in the steady-state, particularly in preparations which lack

Electron Paramagnetic Relaxation and EPR Line Shapes of Manganous Ion Complexes in Aqueous Solutions. Frequency and Ligand Dependence

Line shapes of electron paramagnetic resonance spectra of manganous ion in various ligand environments have been examined at two microwave frequencies, 9.1 and 35 GHz. From the observed differences in line shape at two microwave frequencies and the theoretical expression for relaxation of sextet state ions, the correlation times for processes modulating the zero‐field splitting (zfs) interaction and the magnitude of the effective zfs were evaluated. For all complexes which were examined the correlation times were in the range 3×10−12−9×10−12sec at 300°K. Two types of zfs are distinguished, a transient zfs which is related to the relaxation processes and a static zfs. In this regard there appears to be no simple relation between the transient zfs as obtained from relaxation studies and the static zfs as obtained from solid‐state spectra. The large static zfs for MnEDTA gives quasi‐solid‐state features to the solution EPR spectra of this complex.

Adenylosuccinate synthetase from Azotobacter vinelandii: purification, properties and steady-state kinetics.

Abstract Adenylosuccinate synthetase has been purified to homogeneity from Azotobacter , vinelandii . The purification method involves affinity chromatography on blue dextran-Sepharose, and hydrophobic chromatography, in addition to heat treatment, ammonium sulfate fractionation, and ion-exchange chromatography. The purified enzyme displays a single protein band after electrophoresis in the presence or absence of sodium dodecyl sulfate (SDS). Molecular weights of 110,000 and 54,000 are estimated by gel filtration and SDS gel electrophoresis, respectively. Steady-state kinetic measurements of the forward and reverse reactions and of the reaction in which arsenate replaces phosphate reveal a sequential mechanism with a fully random order of substrate addition in all cases. The maximal velocities of the reverse reaction and arsenolysis are virtually identical, and are approximately 10% of the maximal velocity for the forward reaction. In common with this enzyme from other sources, hadacidin is a potent competitive inhibitor with respect to aspartate ( K i = 0.3 μm ). Specific anions, e.g. nitrate and thiocyanate, are competitive inhibitors with respect to GTP; their effectiveness follows the Hofmeister series. Anion inhibition is synergized by GDP, but binding is exclusive with respect to guanylylimidodiphosphate, suggesting binding of the anions at the site normally occupied by the transferable phosphoryl group of GTP.

Nuclear magnetic resonance studies of the aggregation of dihexanoyllecithin and of diheptanoyllecithin in aqueous solutions

Abstract Aggregation of 1,2-dihexanoyl- sn -glycero-3-phosphocholine (dihexanoyllecithin) and 1,2-diheptanoyl- sn -glycero-3-phosphocholine (diheptanoyllecithin) in aqueous solutions has been investigated by 1 H nuclear magnetic resonance spectroscopy. The chemical shifts and line widths of the NMR signals of the lecithins are dependent on the total concentration of lecithin above the critical micelle concentration. Signals for both lecithins in the aggregated state exhibit line widths which are appreciably smaller than the dipolar line width calculated using the overall rotational correlation time of the micelle. Signals of the α-methylene protons of the carboxylic acid side chains of dihexanoyllecithin and diheptanoyllecithin undergo the greatest change in chemical shift on aggregation. A single averaged spectrum of the α-methylene protons is observed in lecithin solutions of concentrations ranging from one to four times the critical micelle concentration demonstrating that individual lecithin molecules are in rapid exchange, with respect to a frequency of 18 Hz, between the monomeric and the aggregated states. Plots of the chemical shift of the α-methylene protons versus concentration of lecithin approximate a micelle formation curve. At about five times the critical micelle concentration for both dihexanoyllecithin and diheptanoyllecithin the α-methylene pattern indicates that there are at least two magnetic environments for lecithin molecules in the aggregated state. Furthermore, individual lecithin molecules are m slow exchange between the two environments which are distinguished by a chemical shift difference of about 2 Hz.

STRUCTURAL STUDIES OF TRANSITION STATE ANALOG COMPLEXES OF CREATINE KINASE

whereby ATP, hydrolyzed in nervous or contractile processes, may be rapidly replenished from ADP and the “pool” of creatine phosphate. Like most enzymes which utilize ATP as a substrate, creatine kinase requires a divalent cation, e.g., Mg(I1) or Mn(II) , as a cofactor in the catalytic reaction.‘ The use of Mn(I1) as the activator provides the enzyme-substrate complex with the prerequisite paramagnetic reference point for the magnetic resonance experiments reported in this paper. The muscle isozyme of creatine kinase may be crystallized from several sources including rabbit muscle, which is the source of enzyme used in studies reported here. Normally the muscle enzyme exists as a dimer, composed of identical subunits with a total molecular weight of 82,000. Since one reactive sulfhydryl group per monomer has been found to be essential for catalytic activity,‘ the enzyme is assumed to have one active site per subunit. Magnetic resonance studies of creatine kinase spin-labeled at the essential sulfhydryl groups have demonstrated the proximity of these sulfhydryl residues to the active site.3 The reactivity of the essential sulfhydryl groups is substantially altered by the presence of various substrates and combinations of substrates on the enzyme.” In particular, Milner-White and Watts 5 have shown that the essential sulfhydryl groups are completely protected from reaction with sulfhydryl reagents by MgADP and creatine in the presence of specific anions such as nitrate and chloride. These anions also bring about a substantial decrease in the dissociation constants of both creatine and ADP (or metal ADP) from the abortive quaternary complex.s+ Such observations have prompted the suggestion that the anions may occupy the site of the missing phosphoryl group in the abortive quaternary complex. The addition of a specific planar anion to the quaternary metal ADP-creatine-enzyme complex then leads to a structure which resembles that of the transition state of the creatine kinase reaction.5 The present paper reports results of nmr and epr studies of the abortive quaternary complexes formed in the creatine kinase system. Effects of anions

ATP-dependent phosphorylation of α-substituted carboxylic acids catalyzed by pyruvate kinase

Abstract Pyruvate kinase from rabbit muscle catalyzes an ATP-dependent phosphorylation of glycolate to yield 2-phosphoglycolate (F. J. Kayne (1974) Biochem. Biophys. Res. Commun. 59 , 8–13) . An investigation of anologous reactions with other α-substituted carboxylic acids reveals several new substrates for such a phosphorylation reaction. Thus the α-hydroxy carboxylic acids l -lactate, d -lactate, dl -α-hydroxybutyrate, dl -αhydroxyvalerate, l -glycerate, d -glycerate, dl -nitrolactate, and dl -β-chlorolactate are phosphorylated on the α-hydroxy group to give the corresponding phosphoesters. Thioglycolate is also a slow substrate for phosphorylation of the thiol group to give the phosphothioglycolate, and dl -thiolactate is phosphorylated in a very slow reaction to give phosphothiolactate. β-Hydroxypyruvate is a substrate; but, unlike the reaction with pyruvate, with β-hydroxypyruvate the equilibrium for the reaction lies in favor of ADP and the phosphorylated product which appears from 31 P NMR data to be tartronate-semialdehyde-2-phosphate. 31 P NMR spectroscopy has been used to verify the identity of the products for all of the reactions. Steady-state kinetic constants have been obtained for some of the more rapid reactions. The reactions with glycolate, l -glycerate, and β-hydroxypyruvate have k cat values that are close to that for phosphorylation of pyruvate in the reverse of the physiological reaction.

Analysis of EPR powder pattern lineshapes for Mn(II) including third-order perturbation corrections. Applications to Mn(II) complexes with enzymes☆

Abstract Third-order perturbation theory is used to obtain expressions for the energy levels of 55Mn(II)( I = 5 2 ; S = 5 2 ) in the presence of an external magnetic field with axial and rhombic distortions in the crystal field. The expressions are used to stimulate line shapes for the central ( 1 2 ↔− 1 2 ) fine structure transition in the EPR spectra for Mn(II) in complexes with enzymes.

An electron paramagnetic resonance study of bovine α-lactalbumin-metal ion complexes

α-lactalbumin has at least three distinct cation binding regions: a Ca(II)-Gd(III) site, a Cu(II)-Zn(II) site and a VO2+ site as observed from electron paramagnetic resonance (EPR) studies of complexes with the bovine protein. Gadolinium, which bound to the calcium site of the protein with a subnanomolar dissociation constant, yielded EPR spectra at 9.5 GHz (X-band) that exhibited features from g = 8 to g = 2. At 35 GHz (Q-band) the central fine structure transition (Ms = 12 → Ms = −12) gave a well-defined powder pattern. The zero-field splitting was large, as reflected in the second-order splitting of the central fine structure transition of about 1 kG. There was also evidence for additional, low affinity binding site(s) for Gd(III). Addition of either Zn(II) or Al(III) did not affect the amplitudes or positions of the bound Gd(III) EPR spectrum. The Cu(II)-α-lactalbumin complex gave a typical axially symmetric spectrum (g¦¦ = 2.260, gXXX = 2.056, A¦¦ = 171 G) with a partially resolved superhyperfine interaction attributable to at least one directly coordinated nitrogen ligand. Addition of Cu(II) to Gd(III)-α-lactalbumin gave an EPR spectrum that was a superposition of signals from the individual Gd(III)- and Cu(II)-α-LA spectra. The absence of any magnetic interactions in the Gd(III)-Cu(II)-α-lactalbumin species indicated that the two cation sites were more than 10 A apart. On the other hand, addition of Zn(II) to Cu(II)-α-lactalbumin gave a set of EPR lines due to free or loosely bound Cu(II), confirming that the Cu(II) was displaced by zinc. The EPR spectra for VO2+-α-lactalbumin at both X- and Q-band were characteristic of an axially symmetric site. The spectral parameters (g¦¦ = 1.936, gXXX = 1.977, A¦¦ = 174.3 × 10−4cm−1, AXXX = 65.1 × 10−4 cm−1) correlated with those expected for four oxygen ligands in the equatorial plane. Neither Ca(II) nor Zn(II) addition displaced VO2+ from its binding site, suggesting that it binds to a unique site on the protein.

Temperature dependent conformational changes at the active site of pyruvate kinase. A li nuclear magnetic resonance study.

Abstract The interaction of Li + , a weak activator of pyruvate kinase, with substrate and inhibitor complexes of the enzyme has been investigated by magnetic resonance techniques. Proton relaxation rate (PRR) titrations indicate that the dissociation constant of Li + from the ternary enzyme-Mn(II)-phospho enol pyruvate (P-enolpyruvate) complex is 15 m m at 5 °C and 17 m m at 30 °C. The electron paramagnetic resonance spectrum of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex is the superposition of spectra for two distinct species (Reed, G. H., and Cohn, M. (1973) J. Biol. Chem. 248 , 6436–6442). Low temperatures favor the form giving rise to the more nearly isotropic spectrum, whereas high temperatures favor the species giving rise to the anisotropic “K + -like” spectrum. 7 Li nuclear magnetic resonance data are consistent with a model in which the two forms observed by epr correspond to differing Mn(II) to Li(I) distances. The form giving rise to the anisotropic spectrum is characterized by a Mn(II) to Li(I) distance of 4.7 A, and in the more isotropic form this distance is approximately 9 A. The 4.7 A separation of the Mn(II) and Li(I) in the anisotropic form of the complex compares favorably with the 4.9 A separation of Mn(II) and T1(I) (Reuben, J., and Kayne, F. J. (1971) J. Biol. Chem. 246 , 6227–6234) in the P-enolpyruvate complex, although T1 + is a much better activator of the pyruvate kinase reaction. Thus, a change in the distance between the monovalent and divalent cations does not account quantitatively for the lower activation by Li + , inasmuch as more than 50% of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex has the “active” conformation with respect to the separation of the cations and the epr spectrum of the complex. As reported previously (Reed, G. H., and Morgan, S. D. (1974) Biochemistry 13 , 3537–3541), the dissociation constant of oxalate and the epr spectrum for the ternary complex of pyruvate kinase with Mn(II) and oxalate are not influenced by the species of monovalent cation present. The nuclear relaxation rates of Li + are increased in the presence of the ternary oxalate complex, although the separation of the Mn(II) and Li(I) appears to be much greater than for the “anisotropic” form of the P-enolpyruvate complex.