Biomedical subjects

F Polticelli

Publications and source records attributed to F Polticelli.

At least 37 records · Page 2Linked to original sources

PubMed1996

Amino acid sequence of chicken Cu, Zn-containing superoxide dismutase and identification of glutathionyl adducts at exposed cysteine residues.

The copper, zinc-containing superoxide dismutase electromorphs from chicken erythrocytes have been isolated, their complete amino acid sequence determined and the identity of the protein moieties established. All electromorphs are constituted by a polypeptide chain made of 153 amino acid residues, corresponding to a molecular mass of 15,598 Da. Accurate molecular mass determination by electrospray mass spectrometry of the separated electromorphs unequivocally proved that, in the chicken superoxide dismutase, either one or two cysteine residues/subunit are involved in a mixed disulfide with glutathione. The same post-translational modification has been proven to occur in human superoxide dismutase. A different rate of S-thiolation by endogenous glutathione was also demonstrated to be responsible for charge heterogeneity in cells. Effect of this modification on the catalytic and molecular properties of superoxide dismutases, and possible mechanisms for the S-thiolation process, were also investigated and discussed.

Amino Acid Sequence↗

PubMed1996

Identification of the residues responsible for the alkaline inhibition of Cu,Zn superoxide dismutase: a site-directed mutagenesis approach.

The catalytic rate of wild type, two single (Lys 120-->Leu, Lys 134-->Thr), and one double (Lys 120-->Leu-Lys 134-->Thr) mutants of Xenopus laevis B Cu,Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the wild-type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK1) and at pH 11.3 (pK2). Catalytic rate measurements on single and double mutants indicate that pK1 is mainly due to the deprotonation of Lys 120 and Lys 134, with only a minor contribution from other surface basic residues, whereas pK2 is due to titration of the invariant Arg 141, likely coupled to deprotonation of the copper-bound water molecule. Accordingly, Brownian dynamics simulations carried out as a function of pH reproduce well the pH dependence of the catalytic rate, when the experimentally determined pKs are assigned to Lys 120, Lys 134, and Arg 141.

Animals↗

PubMed1996

Effect of Lys-->Arg mutation on the thermal stability of Cu,Zn superoxide dismutase: influence on the monomer-dimer equilibrium.

The thermal stability of two single (K3R, K67R) and one double (K3R-K67R) mutants of Xenopus laevis B Cu, Zn superoxide dismutase has been studied to test Lys --> Arg substitution as an 'electrostatically conservative' strategy to increase protein stability. The K3R mutant displays an increased thermostability with respect to the wild-type enzyme, whilst a decreased stability was observed in the case of the K67R and K3R-K67R mutants. Concentration dependence of the apparent inactivation constant (kapp) of the latter mutants, as compared to that of the wild type enzyme and K3R mutant, indicates that their higher sensitivity to heat inactivation is due to a perturbation of the dimer association. These results are confirmed also by fluorescence anisotropy measurements of the internal probe Tyr149. The possible role of Arg67 in perturbing the dimer dissociation equilibrium toward the monomeric form is discussed.

Animals↗

PubMed1995

Low-temperature optical spectroscopy of cobalt in Cu,Co superoxide dismutase: a structural dynamics study of the solvent-unaccessible metal site.

The temperature dependence (300 to 10 K) of the electronic absorption spectra of the cobalt chromophore in bovine superoxide dismutase (SOD) having the native Zn(II) ion selectivity replaced by Co(II) has been investigated in four different derivatives: Cu(II),Co(II) SOD, N3(-)-Cu(II), Co(II) SOD, Cu(I),Co(II) SOD, and E,Co(II) SOD in which the copper ion has been selectively removed. In the Cu(II),Co(II) SOD, the cobalt spectrum is characterized at room temperature by three bands centered at 18,472, 17,670, and 16,793 cm-1; the low-frequency band is split, at low temperatures, into two components, indicating a lower symmetry contribution to a predominantly tetrahedral crystal field. Addition of N3- to the Cu(II),Co(II) SOD introduces slight changes in all the Co(II) visible bands, indicating the occurrence of minor perturbations of the structural cobalt site upon anion binding to the catalytic copper site. Analysis of the spectra in the Cu(I),Co(II) and E,Co(II) enzymes indicates that the His61 imidazolate bridge is released from the copper upon reduction. This is also confirmed by the analysis of the zeroth, first, and second moments of the various bands in the derivatives. The cobalt site is characterized by a harmonic dynamics, at variance with what observed in the solvent accessible copper site [Cupane, A., Leone, M., Militello, V., Stroppolo, M. E., Polticelli, F., & Desideri, A. (1994) Biochemistry 33, 15103-15109]. The degree of local microheterogeneity at the cobalt site is smaller than that observed for the copper site and increases in the order N3(-)-Cu(II),Co(II) approximately Cu(II),Co(II) < Cu(I),Co(II) < E,Co(II) indicating a different local packing and the presence of different constraints on the cobalt site in the four derivatives. The different dynamic behavior with respect to the catalytic, solvent-accessible, copper site is discussed.

Animals↗

PubMed1995

Identification of the residues responsible for the alkaline inhibition of the activity of Cu,Zn superoxide dismutase: a study of native and chemically modified enzymes.

The pH dependence of the activity of Cu,Zn superoxide dismutases from bovine erythrocytes and shark liver was studied by pulse radiolysis in both the native enzymes and those chemically modified at lysine side chains. The study was aimed at identifying the residues responsible for the activity decrease at pH > 9, observed in all native Cu,Zn superoxide dismutases, and is based on the Lys-->Arg substitution present in the shark protein at position 134, which has been established to be critical for the catalytic efficiency of the enzyme. Both native enzymes display a pH dependence that can be deconvoluted by three deprotonation equilibria, at pH 9-9.5 (pK1), at pH 10.2 (pK2), and at pH 11.5 (pK3). pK1 is lacking in both the modified enzymes and thus can be assigned to activity-linked lysine residues. pK2 is clearly dominated by Arg134 in the modified shark enzyme and can be assigned to surface arginine residues. pK3 is shared by all four enzyme forms and is likely to be due to the invariant Arg141.

Amino Acid Sequence↗

PubMed1995

Modulation of the catalytic rate of Cu,Zn superoxide dismutase in single and double mutants of conserved positively and negatively charged residues.

The catalytic rate of four single and three double mutants of Xenopus laevis Cu,Zn superoxide dismutase B, neutralized at Lys120, Asp130, Glu131, and Lys134, has been determined by pulse radiolysis as a function of ionic strength. Neutralization of Glu131 increases the catalytic rate by 80% at low ionic strength, but the effect is reduced to 50% at physiological ionic strength. The rate is unperturbed upon neutralization of Asp130, while neutralization of either of the two lysines drastically decreases the enzyme activity. The Lys120Leu-Lys134Thr and Lys134Thr-Asp130Gln double mutations have an additive and a compensative effect, respectively, on the activity values, while neutralization of the Glu131-Lys134 pair, which also has a compensative effect, gives rise to a faster enzyme at any ionic strength value. The effects observed in the single Asp130Gln and Lys120Leu mutants differ from those reported on human or bovine enzymes [Getzoff et al. (1992) Nature (London) 358, 347-351; Sines et al. (1990) Biochemistry 29, 9403-9412], indicating that some residues occupying the same position in the linear sequence of different Cu,Zn superoxide dismutases have a different functional weight. Our results also suggest that the strategy of multiple charge mutation may be a promising approach in order to increase the catalytic rate of Cu,Zn SODs independently of ionic strength.

Animals↗

PubMed1995

Modelling the three-dimensional structure and the electrostatic potential field of two Cu,Zn superoxide dismutase variants from tomato leaves.

The three-dimensional structure of tomato P31 and T10 Cu,Zn superoxide dismutases (SODs) were computer modelled using the structure of the bovine enzyme as a template. The structure-essential residues retain in the models the position occupied in the other Cu,Zn SODs of known 3D structure and the overall packing of the beta-barrel is maintained. Formation of 'aromatic pairs' occurs between newly inserted aromatic residues. The number of total charges changes in the two variants and some charged residues located in the proximity of the active site in most Cu,Zn SODs disappear in tomato enzymes. Calculation of the electrostatic potential field, carried out by numerically solving the Poisson-Boltzmann equation, indicates that in both variants a negative potential field surrounds all the protein surface except the active site areas, characterized by positive potential values, as already observed in the bovine enzyme. This result confirms that coordinated mutations of charged residues have occurred in the evolution of this enzyme giving rise to a peculiar electrostatic potential distribution common to all members of this protein family.

Amino Acid Sequence↗

PubMed1994

Low-temperature optical spectroscopy of native and azide-reacted bovine Cu,Zn superoxide dismutase. A structural dynamics study.

The optical absorption spectra of native and N(3-)-reacted Cu,Zn superoxide dismutase (SOD) has been studied in the temperature range 300-10 K. The broad d-d bands observed in the room temperature spectrum, centered at 14,700 cm-1 (native enzyme) and at 15,550 cm-1 (N(3-)-reacted enzyme), are clearly split at low temperature into two bands each, centered at 12,835 and 14,844 cm-1 and at 14,418 and 16,300 cm-1, respectively. The thermal behavior of the 23,720 cm-1 band present in the spectrum of the native enzyme indicates that this band belongs to the His61-->Cu(II) ligand to metal charge transfer transition. Analysis of the zeroth, first, and second moments of the various bands as a function of temperature allowed us to obtain useful information on the stereodynamic properties of the metal site in SOD. In particular for the native protein, it was possible to infer a variation in the metal ligand relative position that occurs as the temperature is lowered and that likely involves all of the ligands except His61. On the other hand, the site is stabilized upon N3- binding, and in this case a variation in the metal ligand position is observed only at the level of the bound anion. The possible relation of these properties to the catalytic mechanism of the enzyme is discussed.

Animals↗

PubMed1994

Mutation of Lys-120 and Lys-134 drastically reduces the catalytic rate of Cu,Zn superoxide dismutase.

Lys-120 and Lys-134, located at the edge of the active site channel in most Cu,Zn superoxide dismutases, have been suggested to play a major role in steering the anionic substrate towards the catalytic copper ion. In this study, mutants of Xenopus laevis Cu,Zn superoxide dismutase have been engineered, with Lys-120 and Lys-134 changed into leucine and threonine, respectively, and their catalytic properties have been investigated by pulse radiolysis. Results obtained demonstrate that both residues decrease the catalytic rate by about 40%, in partial disagreement with previous brownian dynamics calculations, carried out on bovine Cu,Zn superoxide dismutase.

Animals↗

PubMed1994

Crystal structure of the cyanide-inhibited Xenopus laevis Cu,Zn superoxide dismutase at 98 K.

The crystal structure of cyanide-inhibited X. laevis Cu,Zn superoxide dismutase has been studied and refined based on diffraction data collected at 98 K. The final R-factor for the 27,299 reflections in the 10.0-1.7 A resolution range is 0.170. The cyanide anion, which is a competitive inhibitor expected to mimic the superoxide binding mode, binds directly to the active site copper atom, replacing the coordinated water molecule. Moreover, the anion establishes a strong electrostatic interaction with the guanidinium group of the conserved active site residue Arg141. The coordination sphere of Cu2+ is partly altered with respect to the uninhibited enzyme: a displacement of 0.41 A in subunit A, and 0.27 A in subunit B of the dimeric enzyme is observed for the Cu2+ ions. Only two ligands in the Cu2+ coordination sphere (His46 and His118) are significantly affected by cyanide binding, whereas virtually no rearrangement of the Zn2+ ligands is reported.

Animals↗

PubMed1994

Crystallographic study of azide-inhibited bovine Cu,Zn superoxide dismutase.

The crystal structure of azide-inhibited bovine Cu,Zn superoxide dismutase has been studied and refined based on X-ray synchrotron radiation data, in conjunction with difference Fourier and restrained crystallographic refinement techniques. The final R-factor for the 20,756 reflections in the 10.0 to 2.1 A resolution range is 0.166. In both enzyme subunits, the azide anion, which is a competitive inhibitor expected to mimic the superoxide binding mode, is observed directly coordinated to the Cu2+ at the place of the metal-bound water molecule, forming an ion pair with the conserved active site residue Arg141. The coordination sphere of Cu2+ is partly altered with respect to the uninhibited enzyme: a displacement of 0.67 A in subunit A, and 0.37 A in subunit B of the dimeric enzyme is observed for the Cu2+. Only two ligands in the Cu2+ coordination sphere (His46 and His118) are affected by azide binding, whereas virtually no rearrangement of the Zn2+ ligands is reported.

Animals↗

PubMed1994

Molecular modeling and electrostatic potential calculations on chemically modified Cu,Zn superoxide dismutases from Bos taurus and shark Prionace glauca: role of Lys134 in electrostatically steering the substrate to the active site.

The three-dimensional structure of Cu,Zn superoxide dismutase (SOD) from the shark Prionace glauca was homology modeled on the structure of the bovine enzyme used as a template. Shark SOD displays the conservative substitution of one of the residues involved in the electrostatic guidance of superoxide toward the active site of the enzyme, i.e., Lys134, which is replaced by arginine. The association rate, calculated by brownian dynamics simulations incorporating electrostatic terms, and the enzyme reaction rate, measured by the pulse radiolysis method, were found to be identical for both bovine and shark SODs. In order to quantify the role of Lys134 in electrostatically steering the superoxide to the active site, chemical modification of the lysine residues by carbamoylation was carried out on both bovine and shark SODs. Experimental determination of the enzymatic reaction rate indicated that the charge located on the residue 134 gives 19% contribution to the guidance of superoxide anion at low ionic strength. This value matches the association rates predicted by brownian dynamics.

Amino Acid Sequence↗

PubMed1993

Peculiar spectroscopic and kinetic properties of Cys-47 in human placental glutathione transferase. Evidence for an atypical thiolate ion pair near the active site.

Cys-47, the most reactive cysteine in the homodimeric glutathione transferase (EC 2.5.1.18) from human placenta (class Pi), displays peculiar acid base and spectroscopic properties. The thiolate form of this residue is characterized by a sharp UV absorption spectrum centered at 229 nm with an epsilon = 7,500 M-1 cm-1. The dependence of the apparent extinction coefficient on pH indicates that the sulfhydryl group of Cys-47 has a pKa value of 4.2. Moreover the dependence of the reactivity of Cys-47 toward bromopyruvate and iodoacetamide with pH resembles that found for the functional sulfhydryls of thiol proteases, which have very low pKa values and exist mainly as a mercaptide-imidazole ion pair. The apparent pKa value for Cys-47, calculated by this kinetic approach, is in good agreement with that determined spectroscopically. X-ray crystallographic data indicate that the protonated amino group of Lys-54, 4.9 A from the sulfur atom, is probably involved in the deprotonation of Cys-47. Calculation of the electrostatic potential on the sulfur atom of Cys-47 gives a theoretical pKa value of 3.5 for the sulfhydryl group. The simulated neutralization of Lys-54 shifts the pKa value of Cys-47 to a normal value of 9.5. These findings suggest that at physiological pH values, Cys-47 exists as the thiolate ion stabilized by an ion pair formation with the protonated amino group of Lys-54, and this probably accounts for its high reactivity.

Alkylation↗

PubMed1993

Crystallisation and preliminary crystallographic analysis of recombinant Xenopus laevis Cu,Zn superoxide dismutase b.

The recombinant Cu,Zn superoxide dismutase from the South African frog Xenopus laevis, expressed in E. coli, has been crystallized in a form suitable for high resolution crystallographic investigations. The crystals grow from polyethylene glycol solutions, at pH 6.0, 28 degrees C, and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell edges a = 73.33, b = 68.86, c = 59.73 A, one protein dimer (32,000 M(r)) per asymmetric unit. Diffraction data have been collected to 3.0 A resolution, and a molecular replacement solution found for Xenopus laevis superoxide dismutase using the bovine enzyme as search model. The crystallographic R-factor corresponding to this solution is 0.412, in the 15.0-3.0 A resolution range.

Animals↗

PubMed1993

Electrostatic recognition in redox copper proteins: a 1H NMR study of the protonation behavior of His 19 in oxidized and reduced Cu,Zn superoxide dismutase.

The pK shift of the His 19 residue in bovine Cu,Zn superoxide dismutase was carefully measured by nuclear magnetic resonance spectroscopy as a function of the change of the copper oxidation state. The measured pK values were identical within experimental error, at variance with results obtained with other copper proteins under similar conditions. The DelPhi program, based on a macroscopic dielectric model for the electrostatic interactions in proteins, was used to calculate pK shifts, as a function of charge perturbation introduced by metal oxidation, between the oxidized and the reduced enzyme, which are assumed to have the same structure on the basis of previous spectroscopic data. A nice fit with the experimental pK values was obtained protonating the imidazole of the copper-zinc bridging His 61, which is known to release the copper in the reduced enzyme. Protonation of His 61 in the reduced state gives rise to an electrostatic potential distribution around the protein almost identical to that observed in the oxidized one. These results suggest that a major role for the bridging histidine in Cu,Zn superoxide dismutase is to provide identical electrostatic steering of the substrate in the two oxidation states of the enzyme by redox-linked protonation-deprotonation processes. This property is discussed in comparison with the effect of copper reduction on the distribution of the electric field in small blue copper proteins.

Acids↗

PubMed1992

Evolutionary conservativeness of electric field in the Cu,Zn superoxide dismutase active site. Evidence for co-ordinated mutation of charged amino acid residues.

Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast, and the isoenzymes A and B from the amphibian Xenopus laevis. The three-dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template. The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X. laevis isoenzymes. The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases. These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities. This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family.

Animals↗

PubMed1991

Involvement of the copper in the inhibition of Cu,Zn superoxide dismutase activity at high pH.

The alkaline spectroscopic transition of the copper at the active site of Cu,Zn superoxide dismutase has been reexamined by room temperature EPR, in order to correlate it with the inhibition of the enzyme activity at high pH. The EPR transition is governed by a single prototropic equilibrium, with pK values of 11.3 and 11.1 for ox and shark superoxide dismutase, respectively. This result suggests possible contributions of changes of the copper environment to the higher pK of the activity/pH curve. When Arg141 was chemically modified by phenylglyoxal treatment of the ox protein, a lower pK value (10.8) was obtained, indicating that Arg141 is involved in the observed modifications of the EPR spectra.

Animals↗

PubMed1990

The Cu,Zn superoxide dismutase isoenzymes of Xenopus laevis: purification, identification of a heterodimer and differential heat sensitivity.

The three Cu,Zn superoxide dismutase electromorphs of the amphibian Xenopus laevis were purified by an original procedure. N-terminal sequence analysis demonstrated that they are two different homodimers (AA and BB) and a hybrid heterodimer (AB), arising from the co-expression of duplicated genes. The three forms have the same pI, same enzyme activity and EPR spectra, but different heat-sensitivity, form BB being more resistant than form AA, with form AB showing intermediate sensitivity. Thermostability of BB and the control bovine enzyme was enhanced by a tenfold increase in protein concentration. It is suggested that the higher heat sensitivity of the AA isoenzyme is related to the presence of an extra Cys residue and to an easier dissociation of the protein dimer into monomers.

Amino Acid Sequence↗