RESUMO
The X-ray crystal structure of the carboxypeptidase A-L-benzylsuccinate complex has been refined at 2.0 A resolution to a final R-factor of 0.166. One molecule of the inhibitor binds to the enzyme active site. The terminal carboxylate forms a salt link with the guanidinium group of Arg145 and hydrogen bonds with Tyr248 and Asn144. The second carboxylate group binds to the zinc ion in an asymmetric bidentate fashion replacing the water molecule of the native structure. The zinc ion moves 0.5 A from its position in the native structure to accommodate the inhibitor binding. The overall stereochemistry around the zinc can be considered a distorted tetrahedron, although six atoms of the co-ordinated groups lie within 3.0 A from the zinc ion. The key for the strong inhibitory properties of L-benzylsuccinate can be found in its ability both to co-ordinate the zinc and to form a short carboxyl-carboxylate-type hydrogen bond (2.5 A) with Glu270.
Assuntos
Carboxipeptidases/química , Sítios de Ligação , Carboxipeptidases A , Cristalografia , Substâncias Macromoleculares , Modelos Moleculares , Conformação Proteica , Succinatos/química , Zinco/químicaRESUMO
Cu,Zn superoxide dismutase was investigated crystallographically in the reduced form. Co-ordinate errors were estimated by comparing two independently refined models, based on two different data sets. This gave a detailed error estimation as opposed to the standard sigma A and Luzzati plots, which estimate only the overall error. The high quality of the final model, obtained after scaling together the two data sets, combined with the error estimates allowed a detailed analysis of the protein and solvent structures. An automatic procedure for building and refining solvent structure was tested and found to give reproducible results. Contrary to results obtained from spectroscopic studies, the co-ordination of the metal ions in the catalytic site is preserved in the crystal structure of the reduced enzyme, as compared with the crystal structure of the oxidised form. Analysis of the solvent reveals a well-defined chain of closely packed, hydrogen-bonded water molecules filling the active site groove. This structural feature could serve as a hydrogen bond relay for efficient delivery of protons to the active centre. Analysis of electron density suggests that Glu119 is covalently modified. The modification, if originated in vivo, could have a role in the catalytic mechanism and could affect the overall electrostatic field in the active site. There are significant differences between the active sites of the two crystallographically independent monomers. They are explained in terms of local differences in the crystal environment.
Assuntos
Eritrócitos/enzimologia , Superóxido Dismutase/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Solventes/química , Superóxido Dismutase/sangue , TemperaturaRESUMO
The reaction of human serum apotransferrin with titanium(IV) citrate under physiological conditions results in the formation of a specific bis-titanium(IV) transferrin adduct (Ti2Tf hereafter) with two titanium(IV) ions loaded at the iron binding sites. The same specific Ti2Tf complex is formed by reacting apotransferrin with titanium(III) chloride and exposing the sample to air. The derivative thus obtained was characterized by spectroscopic techniques, including absorption, UV difference, circular dichroism and 13C NMR spectroscopies, and shown to be stable within the pH range 5.5-9.0. Surprisingly, the reaction of apoTf with titanium(IV) nitrilotriacetate (NTA) does not lead to formation of appreciable amounts of Ti2Tf, even after long incubation times, although some weak interactions of Ti(IV)-NTA with apoTf are spectroscopically detected. Implications of the present results for a role of transferrin in the uptake, transport and delivery of soluble titanium(IV) compounds under physiological conditions are discussed.
Assuntos
Apoproteínas/metabolismo , beta-Globulinas/metabolismo , Titânio/metabolismo , Transferrina/metabolismo , Transporte Biológico , Dicroísmo Circular , Humanos , Concentração de Íons de Hidrogênio , Íons , Ácido Nitrilotriacético/metabolismo , Ressonância Magnética Nuclear Biomolecular , Solubilidade , Espectrofotometria Ultravioleta , Fatores de Tempo , Titânio/sangue , Titânio/toxicidadeRESUMO
We have employed transneuronal transport to examine the anatomical relationships between the deep cerebellar nuclei and 2 cortical motor areas: the primary motor cortex and the arcuate premotor area (APA). In the same animals, we have also examined the patterns of labeling in the thalamus and the red nucleus to provide evidence for the potential routes of transneuronal transport to the cerebellum. When the appropriate technical procedures were employed, cortical injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) resulted in transneuronal labeling within portions of the contralateral deep cerebellar nuclei. Injections into the primary motor cortex labeled neurons in the dentate and in the 2 subdivisions of the interpositus. Injections into the APA labeled neurons in the dentate and in only the posterior subdivision of the interpositus. In most instances, dentate neurons were more intensely labeled following the cortical injections than interpositus neurons. The transneuronal labeling observed in the dentate nucleus was topographically organized. The dentate region that was labeled following injections into the "arm area" of the APA was caudal and ventral to the dentate region that was labeled following injections into the "arm area" of the primary motor cortex. This observation provides evidence for two "arm areas" in the dentate: one anatomically related to the APA, and the other related to the primary motor cortex. More than one route of transport may be responsible for the labeling of cerebellar neurons. We propose that the labeling observed in the dentate nucleus reflects the pattern of connections in the cerebellothalamocortical pathways that link the dentate with the cerebral cortex. Thus, our observations support the concept proposed by Schell and Strick (J. Neurosci. 4:539-560, '84)--that the cortical targets of the dentate nucleus include both the primary motor cortex and the APA.
Assuntos
Núcleo Arqueado do Hipotálamo/citologia , Cerebelo/citologia , Córtex Motor/citologia , Núcleo Rubro/citologia , Tálamo/citologia , Animais , Núcleos Cerebelares/citologia , Peroxidase do Rábano Silvestre , Macaca nemestrina , Vias Neurais/anatomia & histologia , Conjugado Aglutinina do Germe de Trigo-Peroxidase do Rábano Silvestre , Aglutininas do Germe de TrigoRESUMO
Gold(III) complexes generally exhibit interesting cytotoxic and antitumor properties, but until now, their development has been heavily hampered by their poor stability under physiological conditions. To enhance the stability of the gold(III) center, we prepared a number of gold(III) complexes with multidentate ligands - namely [Au(en)(2)]Cl(3), [Au(dien)Cl]Cl(2), [Au(cyclam)](ClO(4))(2)Cl, [Au(terpy)Cl]Cl(2), and [Au(phen)Cl(2)]Cl - and analyzed their behavior in solution. The solution properties of these complexes were monitored by visible absorption spectroscopy, mass spectrometry, and chloride-selective potentiometric measurements; the electrochemical properties were also studied by cyclic voltammetry and coulometry. Since all the investigated compounds exhibited sufficient stability under physiological conditions, their cytotoxic properties were tested in vitro, via the sulforhodamine B assay, on the representative human ovarian tumor cell line A2780, either sensitive or resistant to cisplatin. In most cases the investigated compounds showed relevant cell-killing properties with IC(50) values falling in the 0.2-10 microM range; noticeably most investigated gold(III) complexes were able to overcome, to a large extent, resistance to cisplatin when tested on the corresponding cisplatin-resistant cell line. The cytotoxic properties of the free ligands were also determined under the same solution conditions. Ethylenediamine, diethylenetriamine, and cyclam were virtually nontoxic (IC(50) values > 100 microM) so that the relevant cytotoxic effects observed for [Au(en)(2)]Cl(3) and [Au(dien)Cl]Cl(2) could be quite unambiguously ascribed to the presence of the gold(III) center. In contrast the phenanthroline and terpyridine ligands turned out to be even more cytotoxic than the corresponding gold(III) complexes rendering the interpretation of the cytotoxicity profiles of the latter complexes less straightforward. The implications of the present findings for the development of novel gold(III) complexes as possible cytotoxic and antitumor drugs are discussed.
Assuntos
Antineoplásicos/síntese química , Ouro , Compostos Organometálicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Eletroquímica , Humanos , Espectrometria de Massas , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Potenciometria , Soluções , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The gold(III) complexes [Au(phen)Cl2]Cl and [Au(dien)Cl]Cl2 were recently shown to exert important cytotoxic effects in vitro on human tumor cell lines. To elucidate the biochemical mechanisms leading to cell death, the effects produced by these gold(III) complexes on the leukemic CCRF-CEM cell line--either sensitive (CCRF-CEM) or resistant to cisplatin (CCRF-CEM/CDDP)--were analyzed in detail by various techniques. For comparison purposes the effects produced by equitoxic concentrations of cisplatin were also analyzed. First, the dependence of the IC50 values of either complex on the incubation time was investigated. Cytotoxicity experiments confirmed that both gold(III) compounds retain their efficacy against the cisplatin-resistant line: only minimal cross-resistance with cisplatin was detected. Notably, [Au(phen)Cl2]Cl is more cytotoxic than [Au(dien)Cl]Cl2, with IC50 values of 7.4 and 6.0 M at 24 and 72 h, respectively, on the resistant line. Results of the COMET assay point out that both gold(III) complexes directly damage nuclear DNA. Remarkably, DNA damage inferred by either gold(III) complex in the two cell lines is larger than that produced by equitoxic cisplatin concentrations. Finally, the effects that either gold(III) complex produces on the cell cycle were investigated by flow cytometry. It was found that both complexes cause only moderate and transient cell cycle perturbations. Larger cell cycle perturbations are induced by equitoxic concentrations of cisplatin. The implications of the present results for the mechanism of action of cytotoxic gold(III) complexes are discussed.
Assuntos
Ciclo Celular/efeitos dos fármacos , Cisplatino/farmacologia , Dano ao DNA , Ouro/farmacologia , Ouro/uso terapêutico , Leucemia/tratamento farmacológico , Antineoplásicos/farmacologia , Linhagem Celular , Ensaio Cometa , DNA , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Modelos Químicos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The theoretical approach to the calculation of the influence of selective binding of small ligands on DNA helix-coil transition has been described in the previous paper (Lando D. Yu., J. Biomol. Struct. Dyn., (1994)). In the present paper that method is used for the study of DNA protonation and deprotonation in acidic and alkaline medium by theoretical analysis of pH effect on DNA heat denaturation. The mechanism of DNA protonation in acidic medium and pK values of nucleotides are well known. It gave us an opportunity to check the theory without any fitting of pK values. A good agreement between experimental and calculated functions Tm(pH) and delta T(pH) (melting temperature and melting range width) obtained for acidic medium proved the validity of the theory. However, for alkaline medium there was not even qualitative agreement when the agreed-upon mechanism of deprotonation was considered. Looking into the cause of the discrepancy, we have studied the DNA melting for different mechanisms of deprotonation by calculation of Tm(pH) and delta T(pH). As a result, it has been established that the discrepancy is due to deprotonation of bonded GC base pairs of helical DNA regions (pK = 11). It was shown that the early known protonation and newly found deprotonation of helical DNA essentially stabilised double helix in alkaline and acidic medium.
Assuntos
DNA Bacteriano/química , DNA/química , Modelos Teóricos , Conformação de Ácido Nucleico , Animais , Bovinos , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Matemática , Termodinâmica , TimoRESUMO
Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.
Assuntos
DNA/química , Porfirinas/química , Dicroísmo Circular , Metais , Ácidos Nucleicos Heteroduplexes , Espectrofotometria Ultravioleta/métodosRESUMO
A series of ternary complexes between adenosine 5'-triphosphoric acid (ATP), 2,2'-bipyridyl, and the transition metal ions manganese (II), cobalt(II), copper (II), and zinc(II) in the ratio 1:1:1 have been prepared. The solid compounds are crystalline and can be formulated as [M(II)-H2ATP-2,2'-Bipyridyl]2 . 4H2O (MATPbipy). X-ray powder patterns show them to be all isomorphous. Potentiometric titrations in aqueous solutions are in agreement with the presence of two ionizable protons. Ultraviolet and visible spectra, epr, and magnetic susceptibility measurements suggest that the metal ions have a high-spin distorted octahedral coordination. From infrared spectra it can be deduced that ATP coordinates to the metal only through the oxygen atoms of the phosphate groups. These compounds, which are particularly stable towards hydrolysis, form possible models for ATP transport in biological fluids.
Assuntos
2,2'-Dipiridil , Trifosfato de Adenosina , Metais , Piridinas , Trifosfato de Adenosina/metabolismo , Transporte Biológico Ativo , Cobalto , Cobre , Manganês , Modelos Químicos , ZincoRESUMO
Formation of adducts between the antitumor ruthenium(III) complex [HInd]trans-[RuCl(4)(Ind)(2)] (KP1019) and the plasma proteins serum albumin and serum transferrin was investigated by UV-vis spectroscopy, for metal-to-protein ratios ranging from 1:1 to 5:1. In both cases, formation of tight metal-protein conjugates was observed. Similar spectroscopic features were observed for both albumin and transferrin derivatives implying a similar binding mode of the ruthenium species to these proteins. Surface histidines are the probable anchoring sites for the bound ruthenium(III) ions in line with previous crystallographic results. In order to assess the stability of the KP1019-protein adducts the influence of pH, reducing agents and chelators was analysed by UV-vis spectroscopy. Notably, there was no effect of addition of EDTA on the UV-vis spectra of the conjugates. The pH-stability was high in the pH range 5-8. Experiments with sodium ascorbate showed that there was just some alteration of selected bands. The implications of the present results are discussed in relation to the pharmacological behavior of this novel class of antitumor compounds.
Assuntos
Antineoplásicos/química , Dimetil Sulfóxido/análogos & derivados , Dimetil Sulfóxido/química , Compostos Organometálicos/química , Soroalbumina Bovina/química , Transferrina/química , Animais , Antineoplásicos/metabolismo , Bovinos , Dimetil Sulfóxido/metabolismo , Compostos Organometálicos/metabolismo , Ligação Proteica , Compostos de Rutênio , Soroalbumina Bovina/metabolismoRESUMO
The reactions with DNA of two antitumor active organotin(IV) compounds, the dimer of bis[(di-n-butyl 3,6-dioxaheptanoato)tin] (C(52)H(108)Sn(4)O(1) x 2H(2)O), compound 1, and tri-n-butyltin 3,6,9-trioxodecanoate (C(19)H(40)SnO(5) x 1/2H(2)O), compound 2, were analysed by circular dichroism, DNA melting experiments and gel mobility shift assays. It is found that both complexes modify only slightly the B-type circular dichroism spectroscopy (CD) spectrum of calf thymus DNA. On the other hand, both complexes were found to affect significantly the parameters of the thermally induced helix-to-coil transition. Addition of 1 or 2 to calf thymus DNA samples does not favor DNA renaturation after melting ruling out formation of interstrand crosslinks. Moreover, the effects of both compounds on plasmid DNA gel mobility were investigated. From the analysis of the present results it is inferred that both organotin(IV) compounds do interact with DNA, probably at the level of the phosphate groups.
Assuntos
Antineoplásicos/farmacologia , DNA/efeitos dos fármacos , Compostos Orgânicos de Estanho/farmacologia , Timo/metabolismo , Animais , Bovinos , Dicroísmo Circular , DNA/química , Conformação de Ácido NucleicoRESUMO
The reactivity in solution of two recently characterized gold(III) complexes, AuCl3(Hpm) and AuCl2(pm), has been investigated in view of their potential use as anti-cancer agents. In water, both compounds undergo relatively fast hydrolysis of the bound chlorides without loss of the heterocycle ligand; the process is much faster within a physiological buffer. When the two gold(III) complexes react with proteins like albumin or transferrin, reduction of gold(III) to gold(I) and/or hydrolysis is observed. On the other hand, both complexes bind rapidly and tightly to either polynucleotides or calf thymus DNA, with gold remaining in the +3 oxidation state. Circular dichroism investigations reveal a large perturbation of DNA conformation upon gold(III) binding; preferential binding to GC sequences is shown. Cytotoxicity studies on a number of tumor cell lines demonstrate a good activity of these gold(III) complexes compared to cisplatin. However, quick hydrolysis and/or reduction of these compounds under physiological conditions may represent a severe limitation to their use.
Assuntos
Antineoplásicos/toxicidade , DNA/química , Compostos de Ouro/farmacologia , Polidesoxirribonucleotídeos/química , Albumina Sérica/química , Transferrina/química , Sobrevivência Celular/efeitos dos fármacos , Cloretos/toxicidade , Cisplatino/toxicidade , DNA/efeitos dos fármacos , Compostos de Ouro/química , Compostos de Ouro/toxicidade , Humanos , Conformação de Ácido Nucleico/efeitos dos fármacos , Polidesoxirribonucleotídeos/metabolismo , Albumina Sérica/metabolismo , Soluções , Espectrofotometria , Transferrina/metabolismo , Células Tumorais CultivadasRESUMO
The chloro glycylhistidinate gold(III) complex (GHAu) is shown to be fairly cytotoxic towards the established A2780 ovarian carcinoma human cell line either sensitive or resistant to cisplatin. Remarkably, GHAu is far more cytotoxic than the corresponding zinc(II), palladium(II), platinum(II) and cobalt(II) complexes implying that cytotoxicity is essentially to be ascribed to the presence of a gold(III) center. Circular dichroism (CD) spectra, atomic absorption measurements and DNA melting profiles suggest that GHAu in vitro is able to bind DNA, the presumed target for several antitumor metal complexes, and to modify its conformation, even if the observed changes are generally small. Implications of these findings for the mechanism of action of cytotoxic gold(III) complexes are discussed.
Assuntos
Antineoplásicos/toxicidade , DNA/metabolismo , Dipeptídeos/toxicidade , Compostos Organometálicos/toxicidade , Neoplasias Ovarianas/tratamento farmacológico , Animais , Antineoplásicos/metabolismo , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Cisplatino/toxicidade , DNA/efeitos dos fármacos , Dipeptídeos/metabolismo , Resistência a Medicamentos , Feminino , Humanos , Compostos Organoáuricos , Compostos Organometálicos/metabolismo , Espectrofotometria Atômica , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The experimental and theoretical analyses of the conformational transitions of DNA-cis-platinum complexes have been carried out. It is shown that at low concentrations of the ligand, the thermodynamic parameters of the helix-coil transition of the complexes are not the result of the local B-->A transition.
Assuntos
Cisplatino/química , Adutos de DNA/química , DNA/química , Conformação de Ácido Nucleico , Animais , Bovinos , Modelos Químicos , TermodinâmicaRESUMO
Temperature-dependent selectivity in nucleophilic additions is affected by the solvent. The inversion temperature (marked with arrows in the graph) that appears in the nonlinear Eyring plots of ln (anti/syn) versus temperature for the addition of butyllithium to an O-protected alpha-hydroxy aldehyde 1 does not depend on nucleophiles (nBuLi (black triangle), tBuLi (*)), but on the solvent. Its value can be obtained from a plot of the (13)C NMR chemical shift of C=O versus temperature. TBDMS=tBuMe(2)Si.
RESUMO
The structure of the carboxypeptidase A complex with the inhibitor (S)-(+)-1-amino-2-phenylethylphosphonic acid has been determined at 0.23 nm resolution. The delta F map shows electron-density peaks both in the S1 and S'1 sites, where the inhibitor molecule can be modeled in two different orientations with approximate 50% occupancy. In the proposed model, the phosphonate group binds to the zinc ion in a monodentate fashion. Other anchoring groups for the inhibitor molecule are Arg127 (hydrogen bonds with the phosphonate oxygen atoms) and Glu270 (hydrogen bond with the amino group in one of the two orientations). A recent spectroscopic investigation of the complex between cobalt(II) carboxypeptidase A and (S)-(+)-1-amino-2-phenylethylphosphonic acid is essentially in agreement with our results.
Assuntos
Carboxipeptidases/metabolismo , Compostos Organofosforados/metabolismo , Sítios de Ligação , Carboxipeptidases A , Elétrons , Estrutura Molecular , Difração de Raios XRESUMO
The X-ray structures of native carboxypeptidase A and of the enzyme-inhibitor complex with L-phenyl lactate have been refined at 1.54 and 1.45 A resolution to R factors of 0.151 and 0.161, respectively. Crystals of the complex were isomorphous with the native crystals (space group P2(1), a = 51.60, b = 60.27, c = 47.25 A, beta = 97.27 degrees ). The high-resolution electron density allowed correction of many side-chain positions in the classical carboxypeptidase A model. This reflects the advantages of the high-quality complete synchrotron data collected with an imaging plate detector. The conformational changes in the active centre of the enzyme upon binding of the inhibitor are restricted to only two residues, Tyr248 and Arg145. L-Phenyl lactate is bound in the S1' pocket and forms hydrogen bonds to Arg145, Glu270 and to the zinc-bound water molecule. The present structure provides an explanation for the higher stability of the complexes with the products of esterolysis in comparison with those of amidolysis. This is consistent with the finding that product release is rate limiting for esters but not for peptides.
RESUMO
DNA represents the primary target for platinum antitumor metal complexes and is the probable target for newly developed cytotoxic gold(III) complexes. To test this hypothesis the reactions with calf thymus DNA of five representative gold(III) complexes--namely [Au(en)(2)]Cl(3), [Au(dien)Cl]Cl(2), [Au(cyclam)](ClO(4))(2)Cl, [Au(terpy)Cl]Cl(2) and [Au(phen)Cl(2)]Cl--were analyzed in vitro through various physicochemical techniques including circular dichroism, absorption spectroscopy, DNA melting, and ultradialysis. It is shown that all tested complexes interact with DNA and modify significantly its solution behavior. The solution conformation of DNA is affected to variable extents by the individual complexes as shown by CD titration experiments. Notably, in all cases, the gold(III) chromophore is not largely perturbed by addition of calf thymus DNA ruling out occurrence of gold(III) reduction. Ultradialysis experiments point out that the binding affinity of the various complexes for the DNA double helix is relatively low; in most cases the gold(III)/DNA interaction is electrostatic in nature and reversible. The implications of these findings for the mechanism of action of antitumor gold(III) complexes are discussed.
Assuntos
DNA/química , Ouro/química , Compostos Organometálicos/química , Animais , Bovinos , Dicroísmo Circular , Adutos de DNA/química , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Espectrofotometria , Temperatura , TimoRESUMO
Reactions of the complexes bisethylendiammine gold(III) and bisethylendiammine platinum(II) with calfthymus DNA were comparatively analysed. Both complexes bind DNA non-covalently most probably on the basis of electrostatic interactions. Binding of either complex at low ratios results into modest modifications of B-type DNA conformations, as detected by CD. Far larger CD alterations are observed at high ratios. The gold(III) chromophore is scarcely perturbed by DNA addition Binding of [Au(en)(2)]Cl(3) to calf thymus DNA is reversed by sodium cyanide. By analogy with the case of [Pt(en)(2)]Cl(2) it is suggested that Auen acts as a minor groove binder.
RESUMO
Molecular dynamics (MD) calculations have been performed on mutants of superoxide dismutase (SOD) on some residues present in the electrostatic loop. These calculations have provided the solution structures for the mutants Thr-137-->Ile and Arg; Lys-136-->Ala; Glu-132-->Gln; Glu-133-->Gln; Glu-132, Glu-133-->Gln-132, Gln-133 and-->Gln-132, Lys-133. The structural and dynamic properties of these mutants have been correlated with the catalytic properties and available spectroscopic data. The water molecule present in the active site close to the copper ion in wild type (WT) SOD is missing in the MD average structure of the Thr-137-->Ile mutant, while this molecule is present in the MD average structures of all the other mutants and of WT SOD. This agrees with the experimental data. This is an important result that shows the validity of our calculations and their ability to reproduce even subtle structural features. Addition of one or more positive charges on the 132 and/or 133 positions does not sizably perturb the structure of the active site channel, while the introduction of a positively charged residue (Arg) on position 137 has a large effect on the structure of the electrostatic loop. Analysis of the MD average structures of these mutants has pointed out that the simple electrostatic effects of charged residues in the channel are not the only factor relevant for enzymatic behavior but that the structure of the electrostatic loop and the location of the charged residues also contribute to the catalytic properties of SOD.