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Biomedical subjects

I Bertini

Publications and source records attributed to I Bertini.

At least 163 records · Page 9Linked to original sources
PubMed1990

Spectroscopic characterization of polyethyleneglycol modified superoxide dismutase: 1H NMR studies on its Cu2Co2 derivative.

Spectroscopic methods have been employed in order to understand the molecular basis of the decrease in enzymatic activity of the antiinflammatory enzyme copper-zinc superoxide dismutase (SOD) following the covalent binding of polyethyleneglycol (PEG) chains to the protein amino-groups. The PEG modification is a general method recently proposed to improve the therapeutic index of enzymes. 1H NMR spectra on the cobalt substituted PEG-modified SOD, Cu2Co2-PEG-SOD, have been recorded. The signals are quite broad with respect to the unmodified enzyme. This has been interpreted on the basis of the effect of molecular weight on the linewidth. The analysis has shown that the histidine hydrogens involved in metal binding at the enzyme active site are the same in both native and PEG-modified SOD. Similarly, circular dichroism and absorption spectra indicate that the overall conformation of the metal clusters is not perturbed upon modification. On the other hand, azide titration shows that the affinity constant of N-3 for SOD is largely reduced upon PEG modification (K = 154 M-1 and 75 M-1 for the native and modified SOD, respectively). These results indicate that the decrease in enzymatic activity upon surface modification with PEG is not caused by a perturbation of the active site geometry, but to a decrease in the channeling of the O2- ion towards the enzyme active site.

Circular Dichroism↗
PubMed1990

The unusual behavior of the inhibitor S(+)(1-amino-2-phenylethyl)phosphonic acid towards carboxypeptidase A.

The molecular (1-Amino-2-phenylethyl)phosphonic acid is shown to inhibit the enzymatic activity of carboxypeptidase A. Through the spectroscopic investigation of the cobalt(II) substituted enzyme we propose that it binds the enzyme in the 1:1 ratio directly at the metal, probably through the phosphate group like phosphate itself. The aromatic group is proposed to sit in the so-called S1 hydrophobic pocket. This is a unique behavior among the inhibitors of the enzyme. The S'1 site is still available in the binary adduct so that a ternary complex can be obtained with molecules like L-Phenylalanine, which enter that site.

Animals↗
PubMed1989

Water in the active cavity of copper/zinc superoxide dismutase. A water 1H-nuclear-magnetic-relaxation-dispersion study.

Water 1H-nuclear-magnetic-relaxation-dispersion (NMRD) measurements of solutions of several copper/zinc superoxide dismutase isoenzymes as well as mutants of the human isoenzyme have been performed in order to monitor the presence of exchangeable water at the copper(II) center. The results have been compared with other spectroscopic features of the various derivatives and with their catalytic efficiency. The decrease in the amount of water in the first coordination sphere, detected through NMRD, parallels, in most cases, the increase in the tetragonal nature of the copper ion. On the other hand, the catalytic activity seems unrelated to the presence of water. Most strikingly, the Ile137 mutant of the human isoenzyme, approximately equal to 90% active, has no water in the copper coordination sphere; this is taken as evidence that the electron transfer is not a water-mediated process. The variation in the pH dependence of NMRD data between the wild-type enzyme and the human Ile137 mutant has been found to parallel the variation in the pH dependence of activity.

Animals↗
PubMed1989

Cyanide and azide behave in a similar fashion versus cuprozinc-superoxide dismutase.

The 1H NMR spectra of the cyanide adduct of Cu2Co2-superoxide dismutase have been remeasured at pH 7.5. The exchange rate of CN- is slow on the NMR time scale. The correlation with the spectrum of the unligated enzyme has been established through saturation-transfer techniques of the system in which 50% of the cyanide adduct is formed and through comparison with the spectrum of a Cu2Co2-superoxide dismutase-CN- sample in which the histidines have been deuterium labeled at the position epsilon 1. The similarities between the spectra of the CN- and N-3 derivatives are stressed, in particular with respect to the removal from copper coordination of the same histidine, assigned as His-46.

Animals↗
PubMed1989

Active-site modification of superoxide dismutase by H2O2 studied through 1H NMR of the cobalt derivatives.

It is known that H2O2 at pH 10, inactivates copper(II)-zinc(II)-SOD although not much information is available on what happens at the ligands coordinated to the two metal ions. We have reinvestigated the system through the electronic and 1H NMR spectra of the cobalt(II) and copper(II)-cobalt(II) derivatives. Such studies indicate that the coordinated residues are maintained although there is evidence of some flexibility of the donor groups. The coordination around copper is slightly more tetragonal. Azide binding to the copper ion does not cause the complete detachment of one of the histidines from the copper coordination sphere, as happens with the untreated enzyme.

Binding Sites↗
PubMed1989

A 1H NMR study of cobalt(II) arsanilazocarboxypeptidase A.

Cobalt(II) arsanilazotyrosine-248 carboxypeptidase A has been characterized through 1H NMR spectroscopy. The ability of the azoenzyme to form binary and ternary complexes with L- and D-phenylalanine and azide has been investigated. Comparison with the 1H NMR results obtained on unmodified cobalt(II) carboxypeptidase provides direct information on the specific effect of the presence of the azo group on the reactivity of the system. Marked differences in the interaction with D-phenylalanine have been observed, and structural inferences are drawn.

Carboxypeptidases↗
PubMed1988

1H NMR spectroscopic characterization of binary and ternary complexes of cobalt(II) carboxypeptidase A with inhibitors.

The binding of L- and D-phenylalanine and carboxylate inhibitors to cobalt(II)-substituted carboxypeptidase A, Co(II)CPD (E), in the presence and absence of pseudohalogens (X = N3-, NCO-, and NCS-) has been studied by 1H NMR spectroscopy. This technique monitors the proton signals of histidine residues bound to cobalt(II) and is therefore sensitive to the interactions of inhibitors that perturb the coordination sphere of the metal. Enzyme-inhibitor complexes, E.I, E.I2, and E.I.X, each with characteristic NMR features, have been identified. Thus, for example, L-Phe binds close to the metal ion to form a 1:1 complex, whereas D-Phe binds stepwise, first to a nonmetal site and then to the metal ion to form a 2:1 complex. Both acetate and phenylacetate also form 2:1 adducts stepwise with the enzyme, but beta-phenylpropionate gives a 2:1 complex without any detectable 1:1 intermediate. N3-, NCO-, and NCS- generate E.I.X ternary complexes directly with Co(II)CPD.L-Phe and indirectly with the D-Phe and carboxylate inhibitor 2:1 complexes by displacing the second moiety from its metal binding site. The NMR data suggest that when the carboxylate group of a substrate or inhibitor binds at the active site, a conformational change occurs that allows a second ligand molecule to bind to the metal ion, altering its coordination sphere and thereby attenuating the bidentate behavior of Glu-72. The 1H NMR signals also reflect alterations in the histidine interactions with the metal upon inhibitor binding. Isotropic shifts in the signals for the C-4 (c) and N protons (a) of one of the histidine ligands are readily observed in all of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1988

An ENDOR study of human and bovine erythrocyte superoxide dismutase: 1H and 14N interactions.

Hyperfine interactions (1H and 14N) with the paramagnetic Cu(II)-site obtained from frozen solutions of human and bovine erythrocyte superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) as well as from their derivatives produced by anion binding (N3-, CN-) and by depletion of the Zn(II) site were studied using electron nuclear double resonance (ENDOR) spectroscopy at about 15 K. Both interactions were found to be identical in human and bovine erythrocyte superoxide dismutase. In all compounds, an anisotropic, exchangeable 1H interaction with a nearly constant coupling value (approximately 3 MHz along g perpendicular ) was observed which is due to either histidine NH- or water protons. Other proton interactions were tentatively assigned to H beta 1 of His-44, H delta 2 of His-46 and to H beta 2 of His-44. Depletion of the Zn(II) site did not alter appreciably the pattern of the proton interactions. The 14N couplings of the native specimen indicated equivalent coordination, whereas Zn(II) depletion and CN- addition were found to produce either some or drastic inequivalences, respectively. For N3- addition to either the native or the Zn(II)-depleted sample only minor effects on the respective 14N coupling pattern were observed.

Algorithms↗
PubMed1988

The cobalt(II)-alkaline phosphatase system at alkaline pH.

The uptake of cobalt(II) ions by apoalkaline phosphatase at pH 8 (the pH optimum for activity) has been investigated by the combined use of electronic and 1H NMR spectroscopies. The presence of fast-relaxing high spin cobalt(II) ions in the active site cavity of the protein induces sizable isotropic shifts of the 1H NMR signals of metal-coordinated protein residues, allowing us to propose a metal uptake pattern by the various metal binding sites both in the presence and in the absence of magnesium ions. In the absence of magnesium the active site is not organized in specific metal binding sites. The first equivalent of cobalt(II) ions per dimer binds in an essentially unspecific and possibly fluxional fashion, giving rise to a six-coordinated chromophore. The second and third equivalents induce the formation of increasing amounts of metal ions pairs, cooperatively arranged into the A and B sites of the same subunit with a five- and six-coordinated geometry, respectively. The fourth and fifth equivalents induce the formation of fully blocked A-B pairs in both subunits. Magnesium shows the property of organizing the metal binding sites, probably through coordination to the C sites. Electronic and 1H NMR titration with Co2+ ions show that the initial amount of fluxional cobalt is smaller than in the absence of magnesium and that A-B pairs are more readily formed. Titration of fully metalated Co4Mg2alkaline phosphatase samples with phosphate confirms binding of only one phosphate per dimer.

Alkaline Phosphatase↗
PubMed1988

Interaction of anions with the active site of carboxypeptidase A.

Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Anions↗
PubMed1988

13C NMR studies of carboxylate inhibitor binding to cobalt(II) carboxypeptidase A.

Both 13C NMR and electronic absorption spectral studies on cobalt(II) carboxypeptidase A in the presence of acetate and phenylacetate provide evidence for two binding sites for each of these agents. The transverse relaxation rate T2-1 for the 13C-enriched carboxyl groups of the inhibitors is significantly increased when bound to the paramagnetic cobalt carboxypeptidase as compared to the diamagnetic zinc enzyme. The acetate concentration dependence of T2p-1 shows two inflections indicative of sequential binding of two inhibitor molecules. The cobalt-13C distances, calculated by means of the Solomon equation, indicate that the second acetate molecule binds directly to the metal ion while the first acetate molecule binds to a protein group at a distance 0.5-0.8 nm for the metal ion, consistent with it binding to one or more of the arginyl residues (Arg-145, Arg-127, or Arg-71). In the case of phenylacetate, perturbation of the cobalt electronic absorption spectrum shows that binding occurs stepwise. 13C NMR distance measurements indicate that one of the two phenylacetates is bound to the metal in the EI2 complex. These binding sites may correspond to those identified previously by kinetic means (one of which is competitive, the other noncompetitive) with peptide binding. The studies further indicate that it should be possible to map the protein interactions of the carbonyl groups of both substrate and noncompetitive inhibitors during catalysis by means of 13C NMR studies with suitably labeled substrates and inhibitors.

Acetates↗
PubMed1987

Circular dichroism and 1H NMR studies of Co2+- and Ni2+-substituted concanavalin A and the lentil and pea lectins.

Visible absorption, circular dichroism (CD) and magnetic circular dichroism spectra have been recorded for the Ca2+-Co2+ derivatives of the lentil (CCoLcH) and pea (CCoPSA) lectins (Co2+ at the S1 sites and Ca2+ at the S2 sites) and shown to be very similar for both proteins. The visible absorption and magnetic circular dichroism spectra indicate similar octahedral geometries for high spin Co2+ at S1 in both proteins, as found in the Ca2+-Co2+ complex of concanavalin A (CCoPL) (Richardson, C. E., and Behnke, W. D. (1976) J. Mol. Biol. 102, 441-451). The visible CD data, however, indicate differences in the environment around S1 of CCoLcH and CCoPSA compared to CCoPL. 1H NMR spectra at 90 MHz of the Co2+ and Ni2+ derivatives of the lectins show a number of isotropically shifted signals which arise from protons in the immediate vicinity of the S1 sites. Analysis of the spectra of the Co2+ derivatives in H2O and D2O has permitted resonance assignments of the side chain ring protons of the coordinated histidine at S1 in the lectins. Differences are observed in the H-D exchange rate of the histidine NH proton at S1 in concanavalin A compared to the lentil and pea lectins. NMR data of the Ni2+-substituted proteins, together with spectra of the Co2+ derivatives, also indicate that the side chains of a carboxylate ligand and of the histidine residue at S1 are positioned differently in concanavalin A than in the other two lectins. These results appear to account, in part, for the differences observed in the visible CD spectra of the Co2+-substituted proteins. In addition, binding of monosaccharides does not significantly perturb the spectra of the lectins. An unusual feature in the 1H NMR spectra of all three Co2+-substituted lectins is the presence of two exchangeable downfield shifted resonances which appear to be associated with the two protons of a slowly exchanging water molecule coordinated to the Ca2+ ion at S2. T1 measurements of CCoLcH have provided an estimation of the distances from the Co2+ ion to these two protons of 3.7 and 4.0 A.

3-O-Methylglucose↗
PubMed1987

The influence of anions and inhibitors on the catalytic metal ion in Co(II)-substituted horse liver alcohol dehydrogenase.

1H-NMR and electronic spectroscopic data are reported for the interaction of the effector molecule imidazole and the inhibitor molecule pyrazole with horse liver alcohol dehydrogenase whose catalytic zinc ions were replaced by Co(II). In addition 13C-NMR and optical data are given for the binding of acetate to this enzyme species. For the binary complex with imidazole an assignment of the protons of the metal-coordinated imidazole has been made and it was found that the rate of exchange of the effector molecule is slow on the NMR time scale. In the presence of NADH which is bound to the open conformation of the binary complex, the most pronounced change is a shift of the beta-CH2 protons of the metal-coordinated cysteine residues which is attributed to hydrogen bonding interactions between the carboxamide group of the nicotinamide moiety with cysteine 46. The 1H-NMR spectra of the binary complex of Co(II)-HLADH with pyrazole show resonances assigned to the protons in the 3- and 4-positions of the bound inhibitor, the NH proton resonance is not detectable. In the ternary complex with pyrazole and NAD+ only the resonances of the beta-CH2 protons (beyond 150 ppm) are changed whereas the protons of histidine 67 and the bound inhibitor are unchanged. The data demonstrate that the coordination environment of the catalytic metal ion is changed very little when the protein changes from the open to the closed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1987

A spectroscopic investigation of cobalt(II) substituted alkaline phosphatase.

The electronic and 1H NMR spectra are reported for the cobalt(II) alkaline phosphatase (EC 3.1.3.1.) system at pH around 6 in the range 0-2 mol of cobalt per mol protein. It is shown that under the present experimental conditions cobalt(II) selectively populates the A sites. Three isotropically shifted NH signals have been detected in the A site that indicate the presence of three histidines in the coordination sphere of cobalt(II). The electronic spectra and the nuclear relaxation properties are consistent with pentacoordination of cobalt(II) in the A site. The finding of reproducible preparation routes for the derivatives, and of appropriate experimental conditions for the observation of their 1H NMR spectra, open new possibilities for the spectroscopic investigation of alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1986

The metal-binding properties of ovotransferrin. An investigation of cobalt(II) derivatives.

Cobalt(II) ovotransferrin bicarbonate and oxalate ternary complexes were prepared and investigated in the pH range 7-10.5. Cobalt(II) provides an excellent and unique spectroscopic probe to monitor subtle structural differences in solution between the two sites of ovotransferrin and to investigate the structural dependence on pH. CD spectroscopy on one side and 1H NMR spectroscopy of isotropically shifted signals on the other are extremely sensitive techniques and are particularly suited for high spin cobalt(II)-containing compounds. In the case of the oxalate derivative the metal-binding ability of the protein is different at the two binding sites and is pH dependent; the CD spectra reveal two different sites, one of which is clearly pH dependent with a pKa of 9.5. On the contrary the bicarbonate analogue does not show any spectral difference between the two sites; both of them change with pH, the pKa being again 9.5. 1H NMR spectra of the oxalate derivatives at pH 7-8 reveal the presence of conformers, the distribution of which depends on the H2O/D2O ratio. Such conformers are not revealed in the bicarbonate system; at pH around 10 the NMR spectra of both systems show inequivalence between the two sites and/or the presence of different conformers for each site. Such differences are discussed in terms of the possible implications in mechanism and function. The overall spectral data are consistent with the donor groups being two histidines, two tyrosines, the synergistic anion, and possibly a solvent molecule.

Animals↗
PubMed1985

Protease susceptibility of zinc- and apo-carboxypeptidase A.

Proteases in preparations of carboxypeptidase A progressively inactivate solutions of the apoenzyme but not the metal-containing enzyme. Free amino acids generated by proteolysis interfere with spectral studies after reconstituting the apoenzyme with cobalt. Purification by affinity chromatography eliminates this effect. Affinity-purified apoenzyme is susceptible to digestion with chymotrypsin but the metalloenzyme is not.

Apoenzymes↗