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11.
Structural analysis of zinc substitutions in the active site of thermolysin. 总被引:5,自引:3,他引:2 下载免费PDF全文
D. R. Holland A. C. Hausrath D. Juers B. W. Matthews 《Protein science : a publication of the Protein Society》1995,4(10):1955-1965
Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution. The structure of the Co(2+)-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd(2+)- and Mn(2+)-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side-chain movements are similar to adjustments that have been observed previously in association with the "hinge-bending" motion that is presumed to occur during catalysis by the zinc endoproteases. In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect. 相似文献
12.
Ethylene-promoted formation of aerenchyma in seedling roots of Zea mays L. under aerated and non-aerated conditions 总被引:5,自引:0,他引:5
Hendrik Konings 《Physiologia plantarum》1982,54(2):119-124
The role of ethylene in the formation of lysigenous cortical cavities (aerenchyma) in seedling roots of Zea mays L. cv. Capella, has been studied under aerated and non-aerated conditions. Passing roots from air to aerated water or from an aerated nutrient solution to a non-aerated solution, promoted cavity formation and was accompanied by an increase of the endogenous ethylene concentration. When the endogenous ethylene concentration of roots in aerated nutrient solutions, which otherwise would not produce much cavities, was enhanced by applying ethylene gas (0.1 and 1.0 μl 1-1 in air) or the ethylene precursor 1-aminocyclopropane-1-car-boxylic acid, cavity formation was promoted. When, on the contrary, the endogenous ethylene concentration of the roots was reduced by adding the inhibitors of ethylene biosynthesis, cobalt ions and aminooxyacetic acid, or when the ethylene action was prevented by silver ions, cavity formation was prevented. It is concluded that endogenous ethylene controls the induction of cavity formation in the roots. 相似文献
13.
The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd. 相似文献
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A sediment sample with high organic matter and trace metal content was suspended in synthetic river water for four weeks under an inert gas atmosphere. Subsequently, the anaerobic suspension was reoxidized by bubbling air through it. The concentrations of dissolved oxygen, sulfide, ferrous iron, manganese, cadmium, cobalt and the pH-value were measured at close time intervals during the anaerobic incubation. The anaerobic suspension was a post-oxic or sub-oxic environment with oxygen and total sulfide concentrations less than 1 µmole 1–1. Concentrations of dissolved ferrous iron and manganese were 50–150 µmole 1–1 and 5–30 µmole 1–1, respectively. The total sulfide concentration was measured using a sensitive voltammetric technique, with a detection limit of 1 nmole 1–1. A sequential extraction procedure was applied to two sediment samples taken at the end of the anaerobic incubation and after one week of reoxidation. The extractions indicated that cadmium was bound in sulfide minerals under post-oxic conditions. Thermodynamic equilibrium calculations revealed that the concentrations of dissolved cobalt in the post-oxic suspension were limited by the precipitation of cobalt sulfide minerals. 相似文献
15.
Artificial cytochromes c have been prepared with Fe(III) and Co(III) tetrasulfonated phthalocyanines in place of heme. Their structure and properties have been investigated by difference spectroscopy, CD, epr, electrophoresis, molecular weight estimation, and potentiometric measurements. The visible absorption spectra show the main peak at 650 nm for the iron compound 685 nm for the cobalt one. It is shown by CD experiments that incorporation of Fe(III)L or Co(III)L into apocytochrome c markedly increases helical content of the protein. Its conformation is, however, significantly altered as compared with the native cytochrome c. The epr and spectroscopic data show that the iron and cobalt phthalocyanine models represent the low spin species with the metal ions in trivalent state. Electrophoresis and molecular weight estimation indicate these complexes to be monomers. Both phthalocyanine complexes have not affinity for additional ligands characteristic for hemoglobin. They react, however, with CO, NO, and CN- when they are reduced with dithionite. Moreover, Co(II)L-apocyt c is able to combine with oxygen suggesting a structural feature in common with the oxygen-carrying heme proteins. Iron(II) complex in the same conditions is oxidized directly to the ferric state. The half-reduction potentials of Fe(III)L-apocyt c and Co(III)L-apocyt c are +374 mV and +320 mV, respectively. These complexes are reduced by cytochrome c and cytochrome c reductase (cytochrome bc1). 相似文献
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Li[Ni0.9Co0.1]O2 (NC90), Li[Ni0.9Co0.05Mn0.05]O2 (NCM90), and Li[Ni0.9Mn0.1]O2 (NM90) cathodes are synthesized for the development of a Co‐free high‐energy‐density cathode. NM90 maintains better cycling stability than the two Co‐containing cathodes, particularly under harsh cycling conditions (a discharge capacity of 236 mAh g?1 with a capacity retention of 88% when cycled at 4.4 V under 30 °C and 93% retention when cycled at 4.3 V under 60 °C after 100 cycles). The reason for the enhanced stability is mainly the ability of NM90 to absorb the strain associated with the abrupt anisotropic lattice contraction/extraction and to suppress the formation of microcracks, in addition to enhanced chemical stability from the increased presence of stable Mn4+. Although the absence of Co deteriorates the rate capability, this can be overcome as the rate capability of the NM90 approaches that of the NCM90 when cycled at 60 °C. The long‐term cycling stability of NM90 is confirmed in a full cell, demonstrating that it is one of the most promising Co‐free cathodes for high‐energy‐density applications. This study not only provides insight into redefining the role of Mn in a Ni‐rich cathode, it also represents a clear breakthrough in achieving a commercially viable Co‐free Ni‐rich layered cathode. 相似文献
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Laura Pancaro Mario Innamorati Ornella Vergnano Gambi Silvia Occhiochiuso 《Plant biosystems》2013,147(6):265-284
Abstract Cobalt, nickel and chromium effects on germination of Alyssum, during after-ripening and aging. Cobalt (2.5–20mM), nickel (2.5–25mM) and chromium (0.2-1mM) effects have been studied in seeds of three species of Alyssum, of which two, A. bertolonii and A. argenteum, endemics to serpentine soils and one, A. nebrodense, closely related to the others but endemic to the Madonie (Sicily) where it grows on limestone. The results, expressed as percentage of germinated seeds (p), Kotowski coefficient of velocity (v) and germination average ratio (pτ), have been subjected to statistical analysis. The effect per mM is generally inhibitory, not significant for cobalt, weak but significant for nickel, more marked and significant for chromium, linearly correlated to concentration. The inhibition reaches its highest level when seeds are ripe, being significantly related to the control germination capacity (pτ). The values of the specific inhibition (effect/mM modditppτ) for all the three species are higher for chromium, which is more toxic than nickel, and increase from A. nebrodense to A. argenteum, showing that of the two serpentine endemics A. bertolonii is more tolerant both to nickel and chromium. 相似文献