Studies of the pH dependence of the formation of binary and ternary complexes with liver alcohol dehydrogenase

The association of the coenzyme NAD+ to liver alcohol dehydrogenase (LADH) is known to be pH dependent, with the binding being linked to the shift in the pK of some group on the protein from a value of 9-10, in the free enzyme, to 7.5-8 in the LADH-NAD+ binary complex. We have further characterized the nature of this linkage between NAD+ binding and proton dissociation by studying the pH dependence (pH range 6-10) of the proton release, delta n, and enthalpy change, delta Ho(app), for formation of both binary (LADH-NAD+) and ternary (LADH-NAD+-I, where I is pyrazole or trifluoroethanol) complexes. The pH dependence of both delta n and delta Ho(app) is found to be consistent with linkage to a single acid dissociating group, whose pK is perturbed from 9.5 to 8.0 upon NAD+ binding and is further perturbed to approximately 6.0 upon ternary complex formation. The apparent enthalpy change for NAD+ binding is endothermic between pH 7 and pH 10, with a maximum at pH 8.5-9.0. The pH dependence of the delta Ho(app) for both binary and ternary complex formation is consistent with a heat of protonation of -7.5 kcal/mol for the coupled acid dissociating group. The intrinsic enthalpy changes for NAD+ binding and NAD+ plus pyrazole binding to LADH are determined to be approximately 0 and -11.0 kcal/mol, respectively. Enthalpy change data are also presented for the binding of the NAD+ analogues adenosine 5'-diphosphoribose and 3-acetylpyridine adenine dinucleotide.     

Raman microspectroscopic study of effects of Na(I) and Mg(II) ions on low pH induced DNA structural changes

In this work a confocal Raman microspectrometer is used to investigate the influence of Na(+) and Mg(2+) ions on the DNA structural changes induced by low pH. Measurements are carried out on calf thymus DNA at neutral pH (7) and pH 3 in the presence of low and high concentrations of Na(+) and Mg(2+) ions, respectively. It is found that low concentrations of Na(+) ions do not protect DNA against binding of H(+). High concentrations of monovalent ions can prevent protonation of the DNA double helix. Our Raman spectra show that low concentrations of Mg(2+) ions partly protect DNA against protonation of cytosine (line at 1262 cm(-1)) but do not protect adenine and guanine N(7) against binding of H(+) (characteristic lines at 1304 and 1488 cm(-1), respectively). High concentrations of Mg(2+) can prevent protonation of cytosine and protonation of adenine (disruption of AT pairs). By analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high magnesium salt protect the N(7) of guanine against protonation. A high salt concentration can prevent protonation of guanine, cytosine, and adenine in DNA. Higher salt concentrations cause less DNA protonation than lower salt concentrations. Magnesium ions are found to be more effective in protecting DNA against binding of H(+) as compared with calcium ions presented in a previous study. Divalent metal cations (Mg(2+), Ca(2+)) are more effective in protecting DNA against protonation than monovalent ions (Na(+)).     

Synthesis of new compartmental amino-phenolic ligands. Basicity, coordination properties towards Cu(II) and Zn(II) ions. A fluorescent chemosensor for H and Zn(II)

The syntheses of three new compartmental ligands are reported. Each ligand shows two 1,4,7-triazaheptane (dien) moieties separated by different rigid aromatic groups. The dien unit is linked to the spacer through its central N-atom, while each aromatic moiety contains two hydroxyl-phenolic functions. The synthetic aspects involved in attaching two dien subunits to an aromatic group containing two hydroxyl functions were explored. Each ligand synthesized can coordinate two metal ions positioned far from each other; the single dinuclear units will be useful as building blocks in new supramolecular aggregates. The basicity and binding properties of one of the synthesized ligands (3,3′-bis[N,N-bis(2-aminoethyl)aminomethyl]-4,4′-dihydroxybiphenyl (L2)) were potentiometrically studied in aqueous solution. L2 was found to behave as a diprotic acid and as a pentaprotic base under the experimental conditions used. L2 forms stable mononuclear and dinuclear complexes with Cu(II) and Zn(II) ions; the mononuclear species show a tendency to dimerize, while the dinuclear ones are predominant in the presence of two equivalents of M(II) ions in solution.Both protonation and the presence of Zn(II) strongly affect the fluorescence emission properties of L2, which can be used as a new chemosensor for H⁺ and Zn(II) ions. L2 exhibits pH-dependent fluorescence and the emission due to the different protonation of L2 and can be ascribed, above all, to the degree of protonation of the 4,4′-biphenol unit; thus, L2 is more emitting at acidic pH values where the aromatic unit is fully protonated. On the contrary, the Zn-dinuclear species are more emitting from neutral to alkaline pH values exhibiting a CHEF effect which reaches its maximum values (seven times those of the free ligand) at pH 9 with the [Zn₂H₋₂L2]²⁺ species, thus highlighting the sensing properties of this new chemosensor towards Zn(II).     

A family of polyamino phenolic macrocyclic ligands. Acid-base and coordination properties towards Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) ions

The acid-base and coordination properties towards Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) of four polyamino-phenol macrocycles 15-hydroxy-3,6,9-triazabicyclo[9.3.1]pentadeca-11,13,1¹⁵-triene L1, 18-hydroxy-3,6,9,12-tetraazabicyclo[12.3.1]octadeca-14,16,1¹⁸-triene L2, 21-hydroxy-3,6,9,12,15-pentaazabicyclo[15.3.1]enaicosa-17,19,1²¹-triene L3 and 24-hydroxy-3,6,9,12,15,18-hexaazabicyclo[18.3.1]tetraicosa-20,22,1²⁴-triene L4 are reported. The protonation and stability constants were determined by means of potentiometric measurements in 0.15 mol dm⁻³ NMe₄Cl aqueous solution at 298.1 K. L1 forms highly unsaturated Co(II), Cu(II), Zn(II) and Cd(II) mononuclear complexes that are prone to give dimeric dinuclear species with [(MH₋₁L1)₂]²⁺ stoichiometry, in solution. L2 forms stable Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) mononuclear complexes that can coordinate external species as OH⁻ anion, giving hydroxylated complexes at alkaline pH. L3 forms stable Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) mononuclear complexes and Co(II), Ni(II), Cu(II) and Zn(II) dinuclear [M₂H₋₁L3]³⁺ species. L4 forms stable mono- and dinuclear Co(II), Cu(II), Zn(II) and Cd(II) complexes, but only mononuclear species with Pb(II). The effect of macrocyclic size is considered in the discussion of results.     

A continuous spectrophotometric assay for the activation of plant NAD kinase by calmodulin, calcium(II), and europium(III) ions.

A continuous spectrophotometric assay has been developed to quantify the calmodulin, calcium(II) ion, and europium(III) ion dependence of the activation of NAD kinase from pea seedlings. Experimental enzyme activation data are compared with the theoretical curves for the binding of calcium(II) ions to the individual calcium binding sites of calmodulin. These results indicate that the binding of three calcium(II) ions is necessary for activation of plant NAD kinase. Further studies demonstrate that europium(III) ions can replace calcium(II) ions in calmodulin with retention of its ability to activate NAD kinase.     

Interactions in solution of calcium(II) and copper(II) with nucleoside monophosphates: a calorimetric study

The thermodynamic parameters enthalpy and entropy of the interaction between calcium(II) or copper(II) with 5′-UMP, 5′-CMP, 5′-AMP, 5′-GMP or 5′-IMP in aqueous solution were determined calorimetrically (ionic strength adjusted to 0.1 with tetramethylammonium bromide) at 25 °C and pH 7 for Ca(II) or pH 3–5 for Cu(II). The experimental conditions were carefully selected to avoid polynuclear complex formation and nucleotide self-stacking. The calorimetric data confirm the tendency toward macrochelation which was indicated by Sigel after very precise potentiometric studies, and which follows the order Cu(II)>Ca(II) for the metal ions and GMP>IMP>AMP>CMP=UMP for the nucleotides. Macrochelate formation for these metal-nucleoside monophosphate complexes is energetically favorable and entropically unfavorable. Received: 13 August 1999 / Accepted: 1 February 2000     

Volume and enthalpy profiles of CO binding to Fe(II) tetrakis-(4-sulfonatophenyl)porphyrin.

The focus of this study is to examine volume and enthalpy profiles of ligand binding associated with CO-Fe(II) tetrakis-(4-sulfonato phenyl)-porphyrin (COFe(II)4SP) in aqueous solution. Temperature dependent photothermal beam deflection was employed to probe the overall enthalpy and volume changes associated with CO-photolysis and recombination. The analysis demonstrates that ligand recombination occurs with a pseudo first order rate constant of (2.5+/-0.2)x10(4) s(-1) (at 25 degrees C) with a corresponding volume decrease of 6+/-1 ml/mol. The activation enthalpy (DeltaH(double dagger)) and volume (DeltaV(double dagger)) change for CO recombination (determined from temperature/pressure dependent transient absorption spectroscopy) are found to be 3.9 kcal/mol and 8.2 ml/mol, respectively. These data are consistent with a mechanism in which photolysis yields a five-coordinate high spin (H(2)O)Fe(II)4SP complex that recombines in a single step to form the low spin (CO)(H(2)O)Fe(II)4SP complex. Base elimination, often associated with CO photolysis from hemes, is not observed in this system. The overall volume changes suggest a transition state with significant high spin character. Furthermore, these results demonstrate the utility of coupling photothermal techniques with variable pressure/temperature transient absorption spectroscopy to probe heme reaction dynamics.     

Crystal structures of the short-chain flavin reductase HpaC from Sulfolobus tokodaii strain 7 in its three states: NAD(P)(+)(-)free, NAD(+)(-)bound, and NADP(+)(-)bound

4-Hydroxyphenylacetate (4-HPA) is oxidized as an energy source by two component enzymes, the large component (HpaB) and the small component (HpaC). HpaB is a 4-HPA monooxygenase that utilizes FADH(2) supplied by a flavin reductase HpaC. We determined the crystal structure of HpaC (ST0723) from the aerobic thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 in its three states [NAD(P)(+)-free, NAD(+)-bound, and NADP(+)-bound]. HpaC exists as a homodimer, and each monomer was found to contain an FMN. HpaC preferred FMN to FAD because there was not enough space to accommodate the AMP moiety of FAD in its flavin-binding site. The most striking difference between the NAD(P)(+)-free and the NAD(+)/NADP(+)-bound structures was observed in the N-terminal helix. The N-terminal helices in the NAD(+)/NADP(+)-bound structures rotated ca. 20 degrees relative to the NAD(P)(+)-free structure. The bound NAD(+) has a compact folded conformation with nearly parallel stacking rings of nicotinamide and adenine. The nicotinamide of NAD(+) stacked the isoalloxazine ring of FMN so that NADH could directly transfer hydride. The bound NADP(+) also had a compact conformation but was bound in a reverse direction, which was not suitable for hydride transfer.     

A thermodynamic investigation of the coordinating ability of N-(2-hydroxy ethyl)ethylenediamine towards copper(II) and nickel(II) in aqueous solution

The heats of ligand protonation and of complex formation of the ligand N-(2-hydroxyethyl)ethylenediamine (Etolen) with Nickel(II) and Copper(II) have been calorimetrically measured at 25°C in 0.5 M NaClO₄ solution; the corresponding entropy values have also been calculated. Thermodynamic parameters associated with protonation of the ligand Etolen are compared to those of similar amine ligands and the effect of the hydroxy group in Etolen has been evaluated. Enthalpy and entropy effects associated with the formation of Nickel(II), and Copper(II) complexes with Etolen are compared to similar terms for complex formation with the ligand 2,2′-diaminoethylether (Oden). Complexes of the latter ligand contain a coordinated etheral oxygen and are used as a comparison to determine the effect of the hydroxyl group in Etolen.The entropy term for formation of the hydroxy complex [Cu(OH)₂Etolen] is considerably larger than those associated with the formation of other analogous complexes. This is due to a probable opening of the chelate branch containing the alcoholic group during hydrolysis.     

The subcellular distribution and properties of aldehyde dehydrogenases in rat liver

1. Kinetic experiments suggested the possible existence of at least two different NAD(+)-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme, designated enzyme I, was exclusively localized in the mitochondria and that another enzyme, designated enzyme II, was localized in both the mitochondria and the microsomal fraction. 2. A NADP(+)-dependent enzyme was also found in the mitochondria and the microsomal fraction and it is suggested that this enzyme is identical with enzyme II. 3. The K(m) for acetaldehyde was apparently less than 10mum for enzyme I and 0.9-1.7mm for enzyme II. The K(m) for NAD(+) was similar for both enzymes (20-30mum). The K(m) for NADP(+) was 2-3mm and for acetaldehyde 0.5-0.7mm for the NADP(+)-dependent activity. 4. The NAD(+)-dependent enzymes show pH optima between 9 and 10. The highest activity was found in pyrophosphate buffer for both enzymes. In phosphate buffer there was a striking difference in activity between the two enzymes. Compared with the activity in pyrophosphate buffer, the activity of enzyme II was uninfluenced, whereas the activity of enzyme I was very low. 5. The results are compared with those of earlier investigations on the distribution of aldehyde dehydrogenase and with the results from purified enzymes from different sources.     

Dipicolinato complexes of cobalt(II), copper(II) and zinc(II) with thiamine dications

Cobalt(II), copper(II) and zinc(II) dipicolinato complexes having thiamine dication with compositions [HT][CoL₂]·5H₂O (1), [HT][CuL₂]·5H₂O (2) and [HT][ZnL₂]·5H₂O (3) (L = dipicolinato anion, T = thiamine cation) are synthesized, characterized by X-ray diffraction and other spectroscopic techniques. The thiamine part in these complexes exists as divalent cations. The second protonation of thiamine takes place at the less crowded nitrogen atom of the pyrimidine ring. These complexes are stabilized by electrostatic and hydrogen-bonding interactions of -N⁺-H and -COO⁻ groups along with crystallized water molecules. The complexes are stable in solution as determined by ¹H NMR and visible study. The complex 1 has two medicinal components which can be easily separated by controlling pH in aqueous medium. It gets decomposed on treatment with sodium hydroxide at pH > 8 to form neutral complex {[Na₂(μ-H₂O)₃(H₂O)₃][CoL₂]·H₂O}₂.     

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.     

Determining the extremes of the cellular NAD(H) level by using an Escherichia coli NAD(+)-auxotrophic mutant

NAD (NAD(+)) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD(+) level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD(+) auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD(+) de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD(+). We then constructed the NAD(+)-auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD(+) biosynthesis in cells harboring the ntt4 gene. The minimal NAD(+) level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD(+), while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD(+) was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD(+) concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.     

Mg(II) binding by bovine prothrombin fragment 1 via equilibrium dialysis and the relative roles of Mg(II) and Ca(II) in blood coagulation

The first direct equilibrium dialysis titration of the blood coagulation protein bovine prothrombin fragment 1 with Mg(II) is presented. Fragment 1 has fewer thermodynamic binding sites for Mg(II) than Ca(II), less overall binding affinity, and significantly less cooperativity. Several nonlinear curve fitting models were tested for describing the binding of fragment 1 with Mg(II), Ca(II), and mixed metal binding data. The Mg(II) data is represented by essentially five equivalent, noninteracting sites; for Ca(II), a model with three tight, cooperative sites and four "loose", equal affinity, noninteracting sites provides the best model. Based on the reported equilibrium dialysis data and in conjunction with other experimental data, a model for the binding of Ca(II) and Mg(II) to bovine prothrombin fragment 1 is proposed. The key difference between the binding of these divalent ions is that Ca(II) apparently causes a specific conformational change reflected by the cooperativity observed in the Ca(II) titration. The binding of Ca(II) ions to the three tight, cooperative sites establishes a conformation that is essential for phospholipid X Ca(II) X protein binding. The filling of the loose sites with Ca(II) ions leads to charge reduction and subsequent phospholipid X Ca(II) X protein complex interaction. Binding of Mg(II) to bovine prothrombin fragment 1 does not yield a complex with the necessary phospholipid-binding conformation. However, Mg(II) is apparently capable of stabilizing the Ca(II) conformation as is observed in the mixed metal ion binding data and the synergism in thrombin formation.     

Recognition of structurally diverse substrates by type II 3-hydroxyacyl-CoA dehydrogenase (HADH II)/amyloid-beta binding alcohol dehydrogenase (ABAD)

Human type II hydroxyacyl-CoA dehydrogenase/amyloid-beta binding alcohol dehydrogenase (HADH II/ABAD) is an oxidoreductase whose salient features include broad substrate specificity, encompassing 3-hydroxyacyl-CoA derivatives, hydroxysteroids, alcohols and beta-hydroxybutyrate, and the capacity to bind amyloid-beta peptide, leading to propagation of amyloid-induced cell stress. In this study, we examine the structure and enzymatic activity of the homologous rat HADH II/ABAD enzyme. We report the crystal structure of rat HADH II/ABAD as a binary complex with its NADH cofactor to 2.0 A resolution, as a ternary complex with NAD(+) and 3-ketobutyrate (acetoacetate) to 1.4 A resolution, and as a ternary complex with NADH and 17 beta-estradiol to 1.7 A resolution. This first crystal structure of an HADH II confirms these enzymes are closely related to the short-chain hydroxysteroid dehydrogenases and differ substantially from the classic, type I 3-hydroxyacyl-CoA dehydrogenases. Binding of the ketobutyrate substrate is accompanied by closure of the active site specificity loop, whereas the steroid substrate does not appear to require closure for binding. Despite the different chemical nature of the two bound substrates, the presentation of chemical groups within the active site of each complex is remarkably similar, allowing a general mechanism for catalytic activity to be proposed. There is a characteristic extension to the active site that is likely to accommodate the CoA moiety of 3-hydroxyacyl-CoA substrates. Rat HADH II/ABAD also binds amyloid-beta (1-40) peptide with a K(D) of 21 nM, which is similar to the interaction exhibited between this peptide and human HADH II/ABAD. These studies provide the first structural insights into HADH II/ABAD interaction with its substrates, and indicate the relevance of the rodent enzyme and associated rodent models for analysis of HADH II/ABAD's physiologic and pathophysiologic properties.     

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.     

Interaction of copper(II) and nickel(II) with a bis-amide ligand functionalized with pyridine moieties: Thermodynamic and spectroscopic studies in aqueous solution

We synthesized a new bis-amide ligand derived from the l(+)-tartaric acid. We then determined its protonation constants and the stability constants of the copper(II) and nickel(II) chelates by potentiometry as well as ESI-MS and UV-Vis spectroscopy. We found that both metal ions are able to induce the deprotonation and the coordination of an amide nitrogen donor atom. In the case of copper complexes, the data show the formation of two major species: Cu₂(L₂H₋₃)⁺ and Cu₂(LH₋₄). EPR and XAS experiments led us to precise the relative structure of these compounds. In Cu₂(L₂H₋₃)⁺, each metal center is coordinated by pyridinic and amidic nitrogen atoms of one ligand and by nitrogen and oxygen atoms from pyridine and hydroxyl moieties from the other one. In Cu₂(LH₋₄), the copper centers are coordinated by pyridinic and amidic nitrogen atoms, as well as a deprotonated hydroxyl group of the ligand. In this latter complex, the lower value of the Cu-Cu distance determined from EXAFS experiments and compared to the one of the solid species likely involve the formation of an exogeneous hydroxyl bridge between the two copper centers. With Ni(II) ions, the only one major species is the mononuclear Ni(LH₋₂) complex, in which Ni(II) is held in an octahedral environment with the metal center chelated by the two pyridinic and the two amidic nitrogen atoms, and two oxygen atoms from water molecules.     

Two macrocyclic pentaaza compounds containing pyridine evaluated as novel chelating agents in copper(II) and nickel(II) overload

Two pentaaza macrocycles containing pyridine in the backbone, namely 3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),14,16-triene ([15]pyN₅), and 3,6,10,13,19-pentaazabicyclo[13.3.1]nonadeca-1(19),15,17-triene ([16]pyN₅), were synthesized in good yields. The acid-base behaviour of these compounds was studied by potentiometry at 298.2 K in aqueous solution and ionic strength 0.10 M in KNO₃. The protonation sequence of [15]pyN₅ was investigated by ¹H NMR titration that also allowed the determination of protonation constants in D₂O. Binding studies of the two ligands with Ca²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Pb²⁺ metal ions were performed under the same experimental conditions. The results showed that all the complexes formed with the 15-membered ligand, particularly those of Cu²⁺ and especially Ni²⁺, are thermodynamically more stable than with the larger macrocycle. Cyclic voltammetric data showed that the copper(II) complexes of the two macrocycles exhibited analogous behaviour, with a single quasi-reversible one-electron transfer reduction process assigned to the Cu(II)/Cu(I) couple. The UV-visible-near IR spectroscopic and magnetic moment data of the nickel(II) complexes in solution indicated a tetragonal distorted coordination geometry for the metal centre. X-band EPR spectra of the copper(II) complexes are consistent with distorted square pyramidal geometries. The crystal structure of [Cu([15]pyN₅)]²⁺ determined by X-ray diffraction showed the copper(II) centre coordinated to all five macrocyclic nitrogen donors in a distorted square pyramidal environment.     

Crystal structure of NAD+-dependent Peptoniphilus asaccharolyticus glutamate dehydrogenase reveals determinants of cofactor specificity

Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of l-glutamate to α-ketoglutarate using NAD(P) as a cofactor. The bacterial enzymes are hexamers and each polypeptide consists of an N-terminal substrate-binding (Domain I) followed by a C-terminal cofactor-binding segment (Domain II). The reaction takes place at the junction of the two domains, which move as rigid bodies and are presumed to narrow the cleft during catalysis. Distinct signature sequences in the nucleotide-binding domain have been linked to NAD(+) vs. NADP(+) specificity, but they are not unambiguous predictors of cofactor preferences. Here, we have determined the crystal structure of NAD(+)-specific Peptoniphilus asaccharolyticus glutamate dehydrogenase in the apo state. The poor quality of native crystals was resolved by derivatization with selenomethionine, and the structure was solved by single-wavelength anomalous diffraction methods. The structure reveals an open catalytic cleft in the absence of substrate and cofactor. Modeling of NAD(+) in Domain II suggests that a hydrophobic pocket and polar residues contribute to nucleotide specificity. Mutagenesis and isothermal titration calorimetry studies of a critical glutamate at the P7 position of the core fingerprint confirms its role in NAD(+) binding. Finally, the cofactor binding site is compared with bacterial and mammalian enzymes to understand how the amino acid sequences and three-dimensional structures may distinguish between NAD(+) vs. NADP(+) recognition.     

Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition

Extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1) by DNA damage is a major cause of caspase-independent cell death in ischemia and inflammation. Here we show that NAD(+) depletion and mitochondrial permeability transition (MPT) are sequential and necessary steps in PARP-1-mediated cell death. Cultured mouse astrocytes were treated with the cytotoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine or 3-morpholinosydnonimine to induce DNA damage and PARP-1 activation. The resulting cell death was preceded by NAD(+) depletion, mitochondrial membrane depolarization, and MPT. Sub-micromolar concentrations of cyclosporin A blocked MPT and cell death, suggesting that MPT is a necessary step linking PARP-1 activation to cell death. In astrocytes, extracellular NAD(+) can raise intracellular NAD(+) concentrations. To determine whether NAD(+) depletion is necessary for PARP-1-induced MPT, NAD(+) was restored to near-normal levels after PARP-1 activation. Restoration of NAD(+) enabled the recovery of mitochondrial membrane potential and blocked both MPT and cell death. Furthermore, both cyclosporin A and NAD(+) blocked translocation of the apoptosis-inducing factor from mitochondria to nuclei, a step previously shown necessary for PARP-1-induced cell death. These results suggest that NAD(+) depletion and MPT are necessary intermediary steps linking PARP-1 activation to AIF translocation and cell death.