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G H Reed

Publications and source records attributed to G H Reed.

At least 73 records · Page 4Linked to original sources
PubMed1986

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

alpha-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 = 1/2----Ms = -1/2) 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)-alpha-lactalbumin complex gave a typical axially symmetric spectrum (g parallel = 2.260, g perpendicular = 2.056, A parallel = 171 G) with a partially resolved superhyperfine interaction attributable to at least one directly coordinated nitrogen ligand. Addition of Cu(II) to Gd(III)-alpha-lactalbumin gave an EPR spectrum that was a superposition of signals from the individual Gd(III)- and Cu(II)-alpha-LA spectra. The absence of any magnetic interactions in the Gd(III)-Cu(II)-alpha-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)-alpha-lactalbumin gave a set of EPR lines due to free or loosely bound Cu(II), confirming that the Cu(II) was displaced by zinc.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Coordination scheme and stereochemical configuration of manganese(II) adenosine 5'-diphosphate at the active site of 3-phosphoglycerate kinase.

The structure of the MnIIADP complex at the active site of 3-phosphoglycerate kinase from yeast has been investigated by electron paramagnetic resonance (EPR) spectroscopy. Inhomogeneous broadening in the EPR signals for Mn(II) resulting from unresolved superhyperfine coupling to 17O regiospecifically incorporated into ADP shows that Mn(II) is coordinated to the alpha- and beta-phosphate groups of ADP at the active site of the enzyme. The EPR pattern for the enzyme-MnIIADP complex is characteristic of a predominantly axially symmetric zero-field splitting tensor. The symmetry and magnitude of the zero-field splitting interaction suggest that there is an additional negatively charged oxygen ligand in the coordination sphere of Mn(II). EPR measurements for solutions of the enzyme-MnIIADP complex in 17O-enriched water indicate that there are also two or three water molecules in the coordination sphere of the metal ion. EPR data for complexes with the two epimers of [alpha-17O]ADP have been used to determine the stereochemical configuration of the MnIIADP complex at the active site. EPR spectra for Mn(II) in the enzymic complex with (Rp)-[alpha-17O]ADP show an inhomogeneous broadening due to superhyperfine coupling with 17O whereas spectra for (Sp)-[alpha-17O]ADP complexes are indistinguishable from those for matched samples with unlabeled ADP. These results show that 3-phosphoglycerate kinase selectivity binds the alpha configuration of the alpha, beta chelate of MnIIADP. Addition of 3-phosphoglycerate to form the dead-end complex (enzyme-MnIIADP-3-phosphoglycerate) does not alter the EPR spectrum, but addition of vanadate to this complex causes marked changes in the spectral parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1985

Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase: electron paramagnetic resonance studies with oxygen-17-labeled ligands.

Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II)ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1984

The stereochemical course of phospho transfer catalyzed by adenylosuccinate synthetase. A reaction pathway via a phosphorylated intermediate with net inversion.

The stereochemical course of phospho transfer in the reaction catalyzed by adenylosuccinate synthetase from rat muscle has been determined with chiral [gamma-17O,18O]GTP gamma S as a substrate. The stereochemical configuration of the product, inorganic thiophosphate, was determined by 31P NMR after the compound was stereospecifically incorporated into ATP beta S. The reaction goes with net inversion of configuration, which is the course for a single phospho transfer, even though 6-phospho-IMP is probably an intermediate on the normal reaction pathway (Liebermann, I. (1956) J. Biol. Chem. 223, 327-339). The breakdown of this intermediate goes by C-O bond cleavage and so is not a true phospho transfer step. Thus, inversion of configuration during the course of this ligase reaction is consistent with a single phospho transfer step in the overall reaction, the formation of the phosphorylated intermediate.

Adenylosuccinate Synthase↗
PubMed1984

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

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 alpha-substituted carboxylic acids reveals several new substrates for such a phosphorylation reaction. Thus the alpha-hydroxy carboxylic acids L-lactate, D-lactate, DL-alpha-hydroxybutyrate, DL-alpha-hydroxyvalerate, L-glycerate, D-glycerate, DL-nitrolactate, and DL-beta-chlorolactate are phosphorylated on the alpha-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. beta-Hydroxypyruvate is a substrate; but, unlike the reaction with pyruvate, with beta-hydroxypyruvate the equilibrium for the reaction lies in favor of ADP and the phosphorylated product which appears from 31P NMR data to be tartronate-semialdehyde-2-phosphate. 31P 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 beta-hydroxypyruvate have kcat values that are close to that for phosphorylation of pyruvate in the reverse of the physiological reaction.

Adenosine Triphosphate↗
PubMed1982

The stereochemical configuration of Mn(II) . ADP at the active site of creatine kinase elucidated by electron paramagnetic resonance with Rp [alpha-17O]ADP and Sp [alpha-17O]ADP.

The stereochemical configuration of the Mn(II) . ADP complex at the active site of creatine kinase has been elucidated by EPR using chirally labeled [alpha-17O]ADP. Superhyperfine coupling between the 17O nucleus and Mn(II) produces a characteristic inhomogeneous broadening of the EPR signals for Mn(II) whenever the 17O is in the first coordination sphere of the metal ion. Previous experiments with ADP regio-specifically labeled with 17O in the phosphate groups had shown that Mn(II) was coordinated to the alpha and beta positions of ADP in transition state analog complexes with creatine kinase that involved enzyme, Mn(II), ADP, creatine, and anions such as formate and thiocyanate (Reed, G. H., and Leyh, T. S. (1980) Biochemistry 19, 5472-5480). The present experiments were initiated to determine the stereochemical configuration of the Mn(II) . ADP complex at the active site of the enzyme. EPR spectra for Mn(II) in transition state analog complexes with formate, thiocyanate, and nitrate as the stabilizing anions show inhomogeneous broadening from 17O in Sp [alpha-17O]ADP whereas the spectra obtained with Rp [alpha-17O]ADP are indistinguishable from those for matched samples with unlabeled ADP. The precision of the measurements indicates that the stereoselectivity of the enzyme for the delta configuration of the alpha, beta chelate of Mn(II) . ADP is greater than 15:1. The delta configuration is also preferred in fully active enzymic complexes involving the equilibrium mixture of substrates.

Adenosine Diphosphate↗
PubMed1982

Electron paramagnetic resonance studies of MN(II) complexes with myosin subfragment 1 and oxygen 17-labeled ligands.

Ligands in the first coordination sphere of Mn(II) in the complex of MnADP with myosin subfragment 1 from rabbit skeletal muscle have been investigated. EPR spectroscopy was used to detect superhyperfine coupling between unpaired electrons of the metal ion and the nuclei of oxygen atoms specifically labeled with oxygen 17. The results show that ADP is a beta-monodentate ligand for Mn(II) and that there are probably two water oxygens directly bound to Mn(II). The inhibitory complex of vanadate with subfragment 1 . MnADP was also investigated. Vanadate-dependent changes in the EPR spectra for enzyme-bound Mn(II) indicate that the coordination sphere of MN(II) changes upon binding of vanadate. ADP remains a beta-monodenate ligand in the complex and experiments with 17O-labeled water indicate that two oxygen atoms originally in water are ligands in the complex. However, the oxygens of vanadate equilibrate with those of water during sample preparation so that one of these ligands may be a vanadate oxygen. Three additional ligands, probably from the protein, are required to complete the sextet of ligands to Mn(II) in both complexes studied.

Adenosine Diphosphate↗
PubMed1981

EPR studies of the Mn(II) complex with elongation factor Tu and GDP Identification of oxygen ligands to Mn(II) by observation of 17O superhyperfine coupling.

The coordination sphere of Mn(II) in the complex with GDP and elongation factor Tu from Escherichia coli has been probed by EPR spectroscopy with 17O-labeled ligands. Inhomogeneous broadening in the EPR signals for Mn(II) due to unresolved superhyperfine coupling to the 17O nucleus was used to identify directly bound oxygen ligands. Results with GDP selectively enriched with 17O either in the alpha-phosphate or in the beta-phosphate revealed that GDP was a beta-monodentate ligand for Mn(II) in the complex with the protein. Results with 17O-enriched water showed that two water molecules are coordinated to the Mn(II). The EPR spectrum for the complex is characteristic of octahedral coordination for Mn(II). Hence, three ligands from the protein are required to complete the sextet of ligands.

Bacterial Proteins↗
PubMed1980

Identification of the six ligands to manganese(II) in transition-state-analogue complexes of creatine kinase: oxygen-17 superhyperfine coupling from selectively labeled ligands.

The complete coordination scheme for Mn(II) in transition-state-analogue complexes with creatine kinase has been determined by electron paramagnetic resonance (EPR) spectroscopy. Perturbations in the EPR spectra for Mn(II) due to superhyperfine coupling to 17O of selectively labeled ligands have been used to identify oxygen ligands in the first coordination sphere of the metal ion. The results show that in the complex of enzyme-MnADP-formate-creatine, Mn(II) is bound to oxygen ligands from both the alpha- and beta-phosphate groups of ADP, to an oxygen from the carboxylate group of formate, and to three water molecules. In the complex with thiocyanate replacing formate as the stabilizing anion, previous infrared experiments [Reed, G. H., Barlow, C. H., & Burns, R. A., Jr. (1978) J. Biol. Chem. 253, 4153-4158] indicated that the nitrogen from thiocyanate was bound to the Mn(II). The magnitudes of the 17O superphyperfine coupling constants from the O- ligands of the ADP phosphate groups and from the formate carboxylate are approximately equal and are larger than that for the water ligands. The symmetry of the zero-field-splitting tensor for Mn(II) indicates that the oxygens from the alpha- and beta-phosphate groups of ADP and the ligand donor atom from the anion occupy mutually cis positions in the octahedral coordination geometry. Water proton relaxation time measurements show that the three water molecules which are bound to Mn(II) are not in free exchange with the bulk solvent. Hence, an enclosed structure at the active site is indicated. The results suggest that for creatine kinase the activating metal ion is bound to all three phosphate groups in the transition state of the reaction.

Adenosine Diphosphate↗