Changes in MM-CK conformational mobility upon formation of the ADP-Mg(2+)-NO(3)(-)-creatine transition state analogue complex as detected by hydrogen/deuterium exchange

In the presence of ADP, Mg(2+), creatine, and the planar nitrate ion, creatine kinase isoenzymes undergo significant structural changes accompanying the formation of a very stable transition state analogue complex (TSAC). We have compared, by using hydrogen/deuterium exchange followed by proteolysis of the labeled enzyme and mass spectrometric analysis of the peptic peptides, the backbone dynamics fluctuations of the free enzyme and those of the TSAC. In most peptides, exchange is not affected by ligand binding, except that observed in seven areas located in or at the entrance to the active site, where some protection is detected. On the basis of a comparison with the three-dimensional structures of free or liganded guanidino kinases, four of these peptides (residues 54-72, 226-234, 287-311, and 315-333) can be considered part of the substrate binding site. The other three (residues 162-186, 193-201, and 202-224) are not directly involved in the binding of substrates and are located in a dynamic domain, which allows the enzyme to properly align the substrates for optimal catalysis.     

Interaction of creatine kinase and hexokinase with the mitochondrial membranes,and self-association of creatine kinase: crosslinking studies

Summary Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes.The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers.Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase.Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu⁺⁺-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization.In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.Abbreviations AK Adenylate Kinase (EC 2.7.4.3) - CKm Mitochondrial Isoenzyme of Creatine Kinase (EC 2.7.3.2) - DMS Dimethyl Suberimidate - DTT Dithiothreitol - EGS Ethylene Glycol bis (succinimidyl succinate) - EGTA Ethylene Glycol bis (aminoethyl ether) - N,N,N,N Tetraacetic acid - G6P Glucose 6 Phosphate, Hepes - N-2 Hydroxyethyl Piperazine N-2 Ethane Sulfonic Acid - HK Hexokinase (EC 2.7.1.1) - MABI methyl 4-Azido Benzoimidate - NaPi Sodium Phosphate - SANPAH N-Succinimidyl 6(4 azido 2 nitrophenylamino) Hexanoate - SDS Sodium Dodecyl Sulfate (sodium lauryl sulfate) - Tris Tris (hydroxymethyl) Aminomethane     

The effect of Zn2+ on Pelodiscus sinensis creatine kinase: unfolding and aggregation studies

We studied the effects of Zn²⁺ on creatine kinase from the Chinese soft-shelled turtle, Pelodiscus sinensis (PSCK). Zn²⁺ inactivated the activity of PSCK (IC₅₀?=?.079?±?.004?mM) following first-order kinetics consistent with multiple phases. The spectrofluorimetry results showed that Zn²⁺ induced significant tertiary structural changes of PSCK with exposure to hydrophobic surfaces and that Zn²⁺ directly induced PSCK aggregation. The addition of osmolytes such as glycine, proline, and liquaemin successfully blocked PSCK aggregation, recovering the conformation and activity of PSCK. We measured the ORF gene sequence of PSCK by rapid amplification of cDNA end and simulated the 3D structure of PSCK. The results of molecular dynamics simulations showed that eight Zn²⁺ bind to PSCK and one Zn²⁺ is predicted to bind in a plausible active site of creatine and ATP. The interaction of Zn²⁺ with the active site could mostly block the activity of PSCK. Our study provides important insight into the action of Zn²⁺ on PSCK as well as more insights into the PSCK folding and ligand-binding mechanisms, which could provide important insight into the metabolic enzymes of P. sinensis.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Structural, spectroscopic and redox studies of a new ruthenium(III) complex with an imidazole-rich tripodal ligand

The present paper describes a new tripodal ligand containing imidazole and pyridine arms and its first cis-[Ru^III(L)(Cl)₂]ClO₄ complex (1). The crystal structure of 1 shows Ru^III in a distorted octahedral geometry, in which two chloride ions, cis-positioned to each other, are coordinated besides the four nitrogen atoms from the tetradentate ligand L. The cyclic voltammogram of 1 exhibits three redox processes at −67, +73 and +200 mV versus SCE, which are attributed to the Ru^III/Ru^II couple in the cis-[Ru^III(L)(Cl)₂]⁺, cis-[Ru^II(L)(H₂O)(Cl)]⁺ and cis-[Ru^II(L)(H₂O)₂]²⁺, respectively. After chemical reduction (Zn(Hg) or Eu^II) only the cis-[Ru^II(L)(H₂O)₂]²⁺ species is observed in the cyclic voltammetry. Complex 1 absorbs at 470 nm (ε=1.4×10³ mol⁻¹ L cm⁻¹), 335 nm (ε=7.9×10³ mol⁻¹ L cm⁻¹), 301 nm (ε=6.7×10³ mol⁻¹ L cm⁻¹) and 264 nm (ε=9.9×10³ mol⁻¹ L cm⁻¹), in water solution (CF₃COOH, 0.01 mol L⁻¹, μ=0.1 mol L⁻¹ with CF₃COONa). Spectroelectrochemical experiments show a decrease of the bands at 335 and 301 nm, which are attributed to LMCT transitions from the chloride to the Ru^III center and the appearance of a broad band at 402 nm ascribed to MLCT transition from the Ru^II center to the pyridine ligand. The lability of the water ligands in the cis-[Ru^II(L)(H₂O)₂]²⁺ species has been investigated using the auxiliary ligand pyrazine. Reactions in the presence of stoichiometric and excess of pyrazine yield the same species, cis-[Ru^II(L)(H₂O)(pz)]²⁺, which exhibits a reversible redox process at 493 mV versus SCE and absorbs at 438 nm (ε=5.1×10³ mol⁻¹ L cm⁻¹) and 394 nm (ε=4.2×10³ mol⁻¹ L cm⁻¹). Experiments performed with a large excess of pyrazine gave a specific rate constant k₁=(2.8±0.5)×10⁻² M⁻¹ s⁻¹, at 25 °C, in CF₃COOH, 0.01 mol L⁻¹, μ=0.1 mol L⁻¹ (with CF₃COONa).     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in $\text{phosphocreatine}\text{creatine}$ ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in $\text{[}\text{phosphocreatine}\text{]}\text{[}\text{creatine}\text{]}$ ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Creatine kinase kinetics,ATP turnover,and cardiac performance in hearts depleted of creatine with the substrate analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% of the creatine substrate analogue β-guanidinopropionic acid for 6–10 weeks. ³¹P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 μmol/g dry wt in guanidinopropionic acid-fed animals but no change in [P_i], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine → ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 μmol/g dry wt per s in control hearts and decreased 4-fold to 2.5–2.8 μmol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2–7.8 μmol/g dry per s. These rates are 1.5–3-times greater than the rate of the phosphocreatine → ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart.     

Isoenzyme-specific interaction of muscle-type creatine kinase with the sarcomeric M-line is mediated by NH(2)-terminal lysine charge-clamps

Creatine kinase (CK) is located in an isoenzyme-specific manner at subcellular sites of energy production and consumption. In muscle cells, the muscle-type CK isoform (MM-CK) specifically interacts with the sarcomeric M-line, while the highly homologous brain-type CK isoform (BB-CK) does not share this property. Sequence comparison revealed two pairs of lysine residues that are highly conserved in M-CK but are not present in B-CK. The role of these lysines in mediating M-line interaction was tested with a set of M-CK and B-CK point mutants and chimeras. We found that all four lysine residues are involved in the isoenzyme-specific M-line interaction, acting pair-wise as strong (K104/K115) and weak interaction sites (K8/K24). An exchange of these lysines in MM-CK led to a loss of M-line binding, whereas the introduction of the very same lysines into BB-CK led to a gain of function by transforming BB-CK into a fully competent M-line-binding protein. The role of the four lysines in MM-CK is discussed within the context of the recently solved x-ray structures of MM-CK and BB-CK.     

One trinuclear copper(II) complex derived from a new Schiff base ligand based on the dianion of 4-chloro-6-(hydroxymethyl)-2-((3-aminopropylimino)methyl)-phenol: Synthesis, structure, spectroscopic and magnetic properties

A linear trinuclear copper(II) complex (1), prepared from a new Schiff base ligand, namely the dianion of 4-chloro-6-(hydroxymethyl)-2-((3-aminopropylimino)methyl)-phenol, was synthesized and characterized in this paper. The X-ray structural study reveals that the geometry of the central Cu2 ion is elongated octahedral and that of the two side Cu(II) ions is distorted square pyramidal. The magnetic susceptibility measurements from 2 to 300 K reveal medium antiferromagnetic interactions between the Cu(II) ions with a J value of −64.6(1) cm⁻¹.     

14-3-3 protein directly interacts with the kinase domain of calcium/calmodulin-dependent protein kinase kinase (CaMKK2)

Background

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca²⁺/calmodulin-dependent kinase (CaMK) family involved in adiposity regulation, glucose homeostasis and cancer. This upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase is inhibited by phosphorylation, which also triggers an association with the scaffolding protein 14-3-3. However, the role of 14-3-3 in the regulation of CaMKK2 remains unknown.

Synthesis, structure, photoluminescence and theoretical studies of an In(III) complex with 2-(2′-hydroxylphenyl)benzoxazole

The synthesis and characterization of [In(pbx)₃] (1) (Hpbx = 2-(2′-hydroxylphenyl)benzoxazole) are presented. The ground and low lying excited electronic states in 1 are studied using density functional theory level (DFT). The optimized geometry is compared to the experimentally observed structure. Time-dependent density functional theory level (TDDFT) is employed to investigate the excited singlet states. The calculated energies of the low lying singlet states in 1 are in considerable agreement with the experimental data. All the low lying transitions are categorized as π → π∗ ligand-to-ligand charge transfer transitions (LLCT) in nature. The emissive state of 1 is assigned as a singlet metal-perturbed π → π∗ ligand-to-ligand charge transfer transition (LLCT).     

Synthesis and crystal structure of a tetranuclear five-coordinated copper(II) complex with Schiff-base of N-(3-carboxylsalicylidene)-4-(2-iminoethyl)-morpholine

A tetranuclear copper(II) complex [Cu₄L₂(CH₃COO)₂(OH)₂]·6H₂O, in which L stands for the dianion of N-(3-carboxylsalicylidene)-4-(2-iminoethyl)morpholine, was synthesized and characterized by elemental analysis, IR, UV-Vis, TGA and X-ray single crystal diffraction. The crystal structure shows that the coordination unit is centrosymmetric with all the Cu(II) ions in square pyramidal coordination geometry. The coordination unit consists of two equivalent parts [Cu₂L(CH₃COO)(OH)], each containing two Cu(II) ions, a tetradentate N₂O₂ Schiff base dianion L²⁻, a CH₃COO⁻, and a OH⁻ anion. In [Cu₂L(CH₃COO)(OH)], the six coordination atoms (N₂O₄) are nearly coplanar, with Cu(1) and Cu(2) enchased in between; the phenolate oxygen and the OH⁻ oxygen as bridging atoms bind the two Cu(II) ions in close proximity; both O₄ around Cu(1) and N₂O₂ around Cu(2) form the basal plane of the coordination square pyramids. The two parts are connected by sharing two μ₃-OH⁻ oxygens and two μ₂-CH₃COO⁻ oxygens from each other, forming four edge-sharing coordination square pyramids around the four Cu(II) ions. A 3D network is formed through hydrogen bonding along a and c axis, and π-π interaction along b axis.     

Synthesis, spectroscopic and X-ray characterization of a copper(II) complex with the Schiff base derived from pyridoxal and aminoguanidine: NMR spectral studies of the ligand

A copper(II) complex with the pyridoxal-aminoguanidine (PL-AG) Schiff base adduct, as an organic compound of the very potent biological activity and promising pharmacological importance in the treatment of diabetic complications, has been prepared and characterized. The X-ray structural analysis of the [CuCl2(PL-AG)] complex showed that it has a distorted pseudo-square-pyramidal (4+1) structure with the tridentate ONN Schiff base in the equatorial plane, with the Cu-O(1), Cu-N(1) and Cu-N(3) bond lengths of 1.917(2)A, 1.930(2)A and 1.984(2)A, respectively. The bond length of the equatorial Cu-Cl(1) is 2.279(1)A, while that of the apical Cu-Cl(2) is 2.792(1)A. Pyridoxal fragment is coordinated in its zwitterionic form. In addition to the X-ray structural analysis, the complex was characterized by IR spectrometric, conductometric and magnetic techniques, and the ligand itself by IR, 1H and 13C NMR spectra.     

Crystal complex structures reveal how substrate is bound in the -4 to the +2 binding sites of Humicola grisea Cel12A

As part of an ongoing enzyme discovery program to investigate the properties and catalytic mechanism of glycoside hydrolase family 12 (GH 12) endoglucanases, a GH family that contains several cellulases that are of interest in industrial applications, we have solved four new crystal structures of wild-type Humicola grisea Cel12A in complexes formed by soaking with cellobiose, cellotetraose, cellopentaose, and a thio-linked cellotetraose derivative (G2SG2). These complex structures allow mapping of the non-covalent interactions between the enzyme and the glucosyl chain bound in subsites -4 to +2 of the enzyme, and shed light on the mechanism and function of GH 12 cellulases. The unhydrolysed cellopentaose and the G2SG2 cello-oligomers span the active site of the catalytically active H.grisea Cel12A enzyme, with the pyranoside bound in subsite -1 displaying a S31 skew boat conformation. After soaking in cellotetraose, the cello-oligomer that is found bound in site -4 to -1 contains a beta-1,3-linkage between the two cellobiose units in the oligomer, which is believed to have been formed by a transglycosylation reaction that has occurred during the ligand soak of the protein crystals. The close fit of this ligand and the binding sites occupied suggest a novel mixed beta-glucanase activity for this enzyme.     

Metal-ion interactions with carbohydrates. Crystal structure and FT-IR study of the SmCl3-ribose complex

A single-crystal of SmCl₃·C₅H₁₀O₅·5H₂O was obtained from methanol-water solution and its structure determined by X-ray. Two forms of the complex as a pair of anomers and related conformers were found in the single-crystal in a disordered state. One ligand is α-d-ribopyranose in the ⁴C₁ conformation and the other one is β-d-ribopyranose. The anomeric ratio is 1:1. Both ligands provide three hydroxyl groups in ax-eq-ax orientation for coordination. The Sm³⁺ ion is nine-coordinated with five Sm-O bonds from water molecules, three Sm-O bonds from hydroxyl groups of the d-ribopyranose and one Sm-Cl bond. The hydroxyl groups, water molecules and chloride ions form an extensive hydrogen-bond network. The IR spectral C-C, O-H, C-O, and C-O-H vibrations were observed to be shifted in the complex and the IR results are in accord with those of X-ray diffraction.     

Tyrosine-phosphorylated extracellular signal--regulated kinase associates with the Golgi complex during G2/M phase of the cell cycle: evidence for regulation of Golgi structure.

Phosphorylation of the extracellular signal-regulated kinases (ERKs) on tyrosine and threonine residues within the TEY tripeptide motif induces ERK activation and targeting of substrates. Although it is recognized that phosphorylation of both residues is required for ERK activation, it is not known if a single phosphorylation of either residue regulates physiological functions. In light of recent evidence indicating that ERK proteins regulate substrate function in the absence of ERK enzymatic activity, we have begun to examine functional roles for partially phosphorylated forms of ERK. Using phosphorylation site--specific ERK antibodies and immunofluorescence, we demonstrate that ERK phosphorylated on the tyrosine residue (pY ERK) within the TEY activation sequence is found constitutively in the nucleus, and localizes to the Golgi complex of cells that are in late G2 or early mitosis of the cell cycle. As cells progress through metaphase and anaphase, pY ERK localization to Golgi vesicles is most evident around the mitotic spindle poles. During telophase, pY ERK associates with newly formed Golgi vesicles but is not found on there after cytokinesis and entry into G1. Increased ERK phosphorylation causes punctate distribution of several Golgi proteins, indicating disruption of the Golgi structure. This observation is reversible by overexpression of a tyrosine phosphorylation--defective ERK mutant, but not by a kinase-inactive ERK2 mutant that is tyrosine phosphorylated. These data provide the first evidence that pY ERK and not ERK kinase activity regulates Golgi structure and may be involved in mitotic Golgi fragmentation and reformation.     

Synthesis, characterization, cytotoxic activity and DNA binding properties of the novel dinuclear cobalt(III) complex with the condensation product of 2-acetylpyridine and malonic acid dihydrazide

A novel dinuclear cobalt(III) complex with the condensation product of 2-acetylpyridine and malonic acid dihydrazide, N′,N′²-bis[(1E)-1-(2-pyridyl)ethylidene]propanedihydrazide was synthesized and characterized by elemental analysis, spectroscopy (NMR and infrared), and X-ray crystal analysis. The complex showed a moderate activity towards Artemia salina. The highest cytotoxic potential of the complex was observed on the epithelial breast cancer (MDA-361) cell line. The investigated complex induced apoptosis, the early apoptotic cells comprising 28.18%, compared to 5.64% of control cells in the same phase. The interaction of the complex with calf thymus DNA (CT-DNA) was monitored by blue shift and hyperchromism in the UV-vis spectra. The observed intrinsic binding constant (K_b = 4.2 × 10⁵ M^− 1) together with structural analysis of the complex indicate the groove binding.     

Crystal structure of an asymmetric complex of pyruvate dehydrogenase kinase 3 with lipoyl domain 2 and its biological implications

A homodimer of pyruvate dehydrogenase kinase (PDHK) is an integral part of pyruvate dehydrogenase complex (PDC) to which it is anchored primarily through the inner lipoyl-bearing domains (L2) of transacetylase component. The catalytic cycle of PDHK and its translocation over the PDC surface is thought to be mediated by the "symmetric" and "asymmetric" modes, in which the PDHK dimer binds to two and one L2-domain(s), respectively. Whereas the structure of the symmetric PDHK/L2 complex was reported, the structural organization and functional role of the asymmetric complex remain obscure. Here, we report the crystal structure of the asymmetric PDHK3/L2 complex that reveals several functionally important features absent from the previous structures. First, the PDHK3 subunits have distinct conformations: one subunit exhibits "open" and the other "closed" configuration of the putative substrate-binding cleft. Second, access to the closed cleft is additionally restricted by local unwinding of the adjacent alpha-helix. Modeling indicates that the target peptide might gain access to the PDHK active center through the open but not through the closed cleft. Third, the ATP-binding loop in one PDHK3 subunit adopts an open conformation, implying that the nucleotide loading into the active site is mediated by the inactive "pre-insertion" binding mode. Altogether our data suggest that the asymmetric complex represents a physiological state in which binding of a single L2-domain activates one of the PDHK protomers while inactivating another. Thus, the L2-domains likely act not only as the structural anchors but also modulate the catalytic cycle of PDHK.