共查询到20条相似文献,搜索用时 31 毫秒
1.
Reaction of carboxypeptidase A crystals with diazotized arsanilic acid uniquely modifies Tyr-248 to form a monazo derivative, which-in solution-forms an intramolecular inner-sphere coordination complex in the active site zinc atom. tarsanilazocarboxypeptidase exhibits spectral properties that are closely similar to those of the model complex, tetrazolylazo-N-carbobenzoxytyrosine Zn2+, with a distinctive maximum at 510 nm. In addition, its circular dichroic spectrum reveals a negative extremum at this wavelength, also characteristic of this complex. Both spectra are exquisitely responsive to pth changes and serve to monitor formation and dissociation of the metal-azophenol complex. Two pKapp at 7.7 and 9.5 delineate the pH range over which the probe characteristics most effectively gauge conformational features of the active center of arsanilazcarboxypeptidase. Other environmental parameters, e.g., substrates and inhibitors, as well as crystallization of the enzyme also critically influence the formation and dissociation of the complex; the response of the probe suggests that they induce conformational movement of the azoTyr-248 residue away from the zinc atom. tthe now available chemical, functional, structural data bearing on the spatial relationships of Tyr-248 and Zn, both thought critical to catalysis, are evaluated, based on spectra of arsanilazo- and nitrocarboxypeptidase crystals and solutions as well as on detailed kinetic analyses of the native enzyme in both physical states and based on the X-ray structure analysis of the native enzyme and its Gly-L-Tyr complex. Collectively all of the data show that the conformation of carboxypeptidase in crystals differs from that in solution. Moreover, reexamination of the original X-ray maps reported in 1968 and thought to preclude a Tyr-248-Zn interaction now leads to the conclusion that in up to 25 per cent of the molecules in the crystals ttyr-248 interacts with the active site zinc atom (W.D. Lipscomb (1973), Proc. Nat. Acad. Sci U.S. 70, 3797). Thus, even in the crystals the enzyme exists in at least two different conformations. In one of these Tyr-248 is near while in the other it is far from the zinc atom. The spectral effects of Gly-L-Tyr and beta-phenylpropionate on solutions of arsanilazo- and of nitrocarboxypeptidase demonstrate that during the catalytic process Tyr-248 moves away from the zinc atom. This implies a mechanistic role for Tyr-248 different from that postulated on the basis of X-ray crystallographic analysis. Indeed, the proximity of ttyr-248 to the zinc atom, when altered by substrates and inhibitor, may reflect certain of the properties characteristic of the entatic, active site. 相似文献
2.
p-Nitrophenyl p'-guanidinobenzoate and methylumbelliferyl p'-guanidinobenzoate, which are active site titrants for trypsin, and p-nitrophenyl p'-dimethylsulfonioacetamidobenzoate and methylumbelliferyl p'-trimethylammoniocinnamate, which are active site titrants for chymotrypsin, are also hydrolyzed by the respective zymogens. Hydrolysis in each case proceeds via the formation of acyl-zymogens. The acylation rates for the zymogens are 10(3)-10(7) times slower than for the enzymes whereas the deacylation rates of acyl-enzymes and acyl-zymogens are comparable. These findings are consistent with the idea that the diminished catalytic activity of these zymogens is due primarily to their distorted substrate binding sites. The circular dichroic spectra of the acyl-enzymes show induced negative ellipticities in the region of absorption of the acyl group, due to binding of the group in an asymmetric environment. The circular dichroic spectra of the acyl-zymogens do not, but conversion of the acyl-zymogens to acyl-enzymes changes the circular dichroic spectra to those characteristic of the acyl-enzymes. alpha-Carbamyl-epsilon-guanidinated trypsin is a derivative which resembles trypsinogen in lacking activity toward specific ester substrates but possessing low activity toward p-nitrophenyl p'-guanidinobenzoate. The circular dichroic spectrum of the acyl-enzyme formed during hydrolysis of p-nitrophenyl p'-guanidinobenzoate by this derivative resembles that of guanidinobenzoyltrypsinogen, and not that of guanidinobenzoyltrypsin. These circular dichroism studies confirm that the same serine residue is involved in catalysis by both enzymes and zymogens. They demonstrate directly that the acylating group is in a different environment in each and indicate that this specific environment is a determinant in the catalytic activity of each. Thus the circular dichroic spectra of these acyl intermediates provide a sensitive probe of the subtle conformational changes which occur on zymogen activation. The results support the previous conclusion that the major feature of the activation of trypsinogen and chymotrypsinogen is the rearrangement of the substrate binding site and that the appearance of a new amino terminus causes this rearrangement. 相似文献
3.
Circular dichroism and absorption spectra are measured on mixed solutions of acridine orange and poly(S-carboxymethyl-L -cysteine) at different pH and P/D mixing ratios. The observed circular dichroism spectra are classified into several types, mainly based on the number and sign of circular dichroic bands in the visible region. Three of them are associated with the absorption spectra characteristic of dimeric dye or higher aggregates of dye. Type I is observed with solutions, of which the pH is acid and P/D is higher than 4, and it has an unsymmetrical pair of positive and negative dichroic bands at 470 and 430 nm. This type is induced on the dye bound to the polymer in the β-conformation. Types II and III are considered to be characteristic of randomly coiled polymers. Type II is exhibited by solutions of P/D higher than 1 at pH 5–7 and has two dichroic bands around the same wavelengths as Type I but with opposite signs and an additional positive band at 560 nm. Type III, shown by solutions of P/D 2–0.6 at pH 6–10.5, has three dichroic bands around the same wavelengths as Type II but with signs opposite to it. The other two types of circular dichroism, induced for the solutions of P/D less than 1 at slightly acid pH, are associated with the absorption spectra of monomeric dye and are observed with disordered or randomly coiled polymer. They have a pair of dichroic bands at 540 and 425 nm, and the signs of these bands are opposite to each other in these two types. 相似文献
4.
Ahmadi SM Dehghan G Hosseinpourfeizi MA Dolatabadi JE Kashanian S 《DNA and cell biology》2011,30(7):517-523
DNA binding studies of flavonoids are needed to understand the reaction mechanism and improve drugs that target DNA. Quercetin (Q) is one of the most common flavonoids that can chelate metal ions and interact with double-stranded DNA. In the present work, UV absorption spectrophotometry, viscosimetry, circular dichroism, and fluorescence spectroscopic techniques were employed to study the interaction of water-soluble quercetin--molybdenum(VI) complex [Q-Mo(VI)] with calf thymus DNA. The binding constants (K(b)) for the complex with DNA were estimated to be 2.9?×?10(3) through spectroscopic titrations. Upon addition of the complex, significant decreases were observed in the viscosity of calf thymus DNA. Circular dichroic spectra indicated that there are certain detectable conformational changes in the DNA double helix when complex was added. Further, competitive methylene blue binding studies with fluorescence spectroscopy have shown that the complex can bind to DNA through nonintercalative mode. The experimental results suggest that Q-Mo(VI) binds to DNA via an outside binding mode. 相似文献
5.
Phototransformation Pt to Pfr was investigated with 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa L. cv. Pirol) using circular dichroism spectroscopy at -110°C to +30°C. Using absorption spectra of the intermediates formed at the respective temperatures, circular dichroism spectra (300–800 nm) of pure intermediates were calculated.
The sign of the circular dichroic absorption bands changed upon formation of lumi-R, the primary photoproduct of Pr . This would be compatible with a Z→E isomerization taking place at this reaction step. The subsequent intermediates (meta-Ra and meta-Rc ) as well as Pfr showed only small circular dichroism. Their absorption spectra were drastically shifted, but had similar spectral shapes. The results are discussed in terms of conformational changes of the phytochrome chromophore presumably taking place at the early steps of phototransformation Pr to Pfr . 相似文献
The sign of the circular dichroic absorption bands changed upon formation of lumi-R, the primary photoproduct of P
6.
Zakomirdina LN Kulikova VV Gogoleva OI Dementieva IS Faleev NG Demidkina TV 《Biochemistry. Biokhimii?a》2002,67(10):1189-1196
An efficient method for purification of recombinant tryptophanase from Proteus vulgaris was developed. Catalytic properties of the enzyme in reactions with L-tryptophan and some other substrates as well as competitive inhibition by various amino acids in the reaction with S-o-nitrophenyl-L-cysteine were studied. Absorption and circular dichroism spectra of holotryptophanase and its complexes with characteristic inhibitors modeling the structure of the principal reaction intermediates were examined. Kinetic and spectral properties of two tryptophanases which markedly differ in their primary structures are compared. It was found that although the spectral properties of the holoenzymes and their complexes with amino acid inhibitors are different, the principal kinetic properties of the enzymes from Proteus vulgaris and Escherichia coli are analogous. This indicates structural similarity of their active sites. 相似文献
7.
W Mori O Yamauchi Y Nakao A Nakahara 《Biochemical and biophysical research communications》1975,66(2):725-730
The absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectra in the visible region have been measured for hemocyanin at 77, 198, and 293K. From the temperature dependence of the CD spectra of oxyhemocyanin, the bands observed at 450, 565, and 700 nm were resolved into those centered at 430, 490, 565, 600, and 700 nm. Since these five peaks are most probably due to the d-d transitions, the two copper ions at the oxygenated active center are inferred to be Cu(II) ions each in a non-equivalent coordination geometry of very low symmetry. The MCD spectral data confirm the view and reasonably explain the diamagnetism of oxyhemocyanin. 相似文献
8.
This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme. 相似文献
9.
A systematic investigation has been made into the circular dichroic behavior of luteinizing hormone releasing hormone and its peptide fragments and deletion analogues. The results are interpreted to mean that the hormone exists in solution as an ensemble of conformers with different sensitivities to temperature and solvent composition. The far-ultraviolet circular dichroic spectra exhibited by the hormone under different experimental conditions can be simulated satisfactorily by the weighted addition of the spectra of its aliphatic- and aromatic-containing halves. However, the structure of the hormone is not simply the sum of its halves, since some conformational feature of the intact molecule perturbs the near-ultraviolet circular dichroism of its aromatic residues. 相似文献
10.
We have investigated the circular dichroism induced in Cibacron Blue and Congo Red upon binding to several dehydrogenases to probe the conformation of the bound dyes. The circular dichroism spectra of Congo Red are quite similar when the dye is bound to lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase but has bands of opposite sign when bound to cytoplasmic malic dehydrogenase. The circular dichroism spectra of Cibacron Blue bound to these same dehydrogenases are quite different from one another. Since circular dichroism is sensitive to the conformation of bound dye, these differences argue for at least local changes in dye conformation or environment when bound to different dehydrogenases. Congo Red appears to be less sensitive to these effects than Cibacron Blue. 相似文献
11.
The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA. The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA, but depends on the sequence of base pairs. On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300-500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425-440nm and 340-360nm with the maximum varying depending on base composition and sequence of DNA. While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented. 相似文献
12.
Abstract The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA but depends on the sequence of base pairs. On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300–500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425–440nm and 340–360nm with the maximum varying depending on base composition and sequence of DNA While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented. 相似文献
13.
Circular dichroism of holo- and apoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa. 总被引:3,自引:0,他引:3
C T Hou 《Biochemistry》1975,14(17):3899-3902
Circular dichroism studies have been carried out on both apo- and holoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa, in the absence and presence of competitive inhibitors, protocatechualdehyde and 4-nitrocatechol. The apo- and holoenzyme showed identical spectra in the ultraviolet region between 200 and 250 nm (peptide back bone region), but the low intensity negative bands at 330 and 480 nm of the holoenzyme were completely absent in the apoenzyme. On the side chain region, the positive ellipticity peaks of the holoenzyme change into a lower intensity and broader band indicating the participation of aromatic amino acid residues in the primary binding of iron ion. Under anaerobic conditions, spectral changes were evident in the side chain region for the binary complexes of both the holo- and the apoenzyme with protocatechuate. The presence of iron in the holoenzyme results in an increase in positive ellipticity between 290 and 320 nm. Either with or without the iron, the enzyme protein binds protocatechuate and has a greater positive circular dichroism increase at 240-260 nm. CD difference spectra indicate that the modes of binding to form the binary complexes of holo- or apoenzyme with either substrates or competitive inhibitors are different. The bound iron ion stimulates binding. Spectral changes of the holoenzyme in the aromatic region were also observed in different pH environments of lower enzymatic activity. It is still not established whether these aromatic residues play an active or passive role in the binding of iron and/or substrates and inhibitors. 相似文献
14.
Interactions between folate derivatives and palladium(II) complexes are monitored in 1 : 1 molar ratio mixtures by circular dichroic spectra. The results are consistent with the following conclusions, i.e., tetrahydrofolate forms a chelate complex with palladium(II) through nitrogens 5 and 10 which is characterized by a unique circular dichroic spectrum. Mixtures of pallodium (II) complexes and dihydrofolate on methylenetetrahydrofolate could not be expected to, nor do they, give rise to similar circualr dichroic spectra for the following reasons: (1) The N5, N10 chelation site of tetrahydrofolate is blocked in methylenetetrahydrofolate, so coordination of a palladium(II) species must occur at some secondary site. (2) The N5, N10 chelation site is available but carbon six of the pteridine ring system is no longer asymmetric in dihydrofolate. Mixtures of dihdrofolate and palladium(II) complexes have no measurable circular dichroic spectra under the experimental conditions used. 相似文献
15.
T Nishiya I Tabushi A Maeda 《Biochemical and biophysical research communications》1987,144(2):836-840
Delipidated bacteriorhodopsin purified from purple membrane of H. halobium was reconstituted with the circular dichroism active phospholipid. The observed circular dichroism spectra in the 450-700 nm region characteristic of bacteriorhodopsin showed the temperature dependence characterized by a midpoint at ca. 45 degrees C and this spectral change showed the disaggregation of bacteriorhodopsin trimer to monomer. The circular dichroism spectra in the 250-400 nm region characteristic of the azo chromophore of phospholipid exhibited a remarkable temperature dependence synchronized with the disaggregation of bacteriorhodopsin, suggesting that a large proportion of the phospholipid is present as boundary lipid. 相似文献
16.
Zinc ion (Zn(2+)) can be coordinated with four or three amino acid residues to stabilize a protein's structure or to form a catalytic active center. We used phage display selection of a dodecamer random peptide library with Zn(2+) to identify structural zinc sites. The binding specificity for Zn(2+) of selected sequences was confirmed using enzyme-linked immunosorbent and competitive inhibition assays. Circular dichroism spectra indicated that the interaction with Zn(2+) induced a change in conformation, which means the peptide acts as a structural zinc site. Furthermore, a search of protein databases revealed that two selected sequences corresponded to parts of natural zinc sites of copper/zinc superoxide dismutase and zinc-containing ferredoxin. We demonstrated that Zn(2+)-binding sequences selected from the random combinatorial library would be candidates for artificial structural zinc sites. 相似文献
17.
An active derivative of staphylococcal nuclease, in which only tyrosine residue 115 has been nitrated with use of tetranitromethane, has been characterized using absorbance, circular dichroism, and fluorescence spectroscopy. The results show that nitrotyrosine-115 nuclease is indistinguishable from native nuclease with regard to the average secondary structure of the folded polypeptide chain, the susceptibility of the enzyme to heat denaturation, and the local tertiary structure around tryptophan residue 140. Inasmuch as optical properties of nitrotyrosine-115 nuclease from 300 to 500 nm can be unambiguously assigned to nitrotyrosine residue 115 in the active site region, this modified enzyme presents a good model system for studying the circular dichroism properties of this aromatic amino acid in a protein. The spectral properties of nitrotyrosine-115 nuclease have been compared to those of the model compounds, cyclo-(-Gly-Tyr(3NO2)-) and Tyr(3NO2). Circular dichroism spectral changes in nitrotyrosine-115 nuclease due to the binding of deoxythymidine 3',5'-diphosphate and Ca-2+ have been compared to the corresponding nitrotyrosyl-115 absorption spectral changes. This comparison shows that the circular dichroism difference spectrum exhibits an over-all change in the intensity of the observed Cotton effects, whereas the absorption difference spectrum exhibits a blue shift. This finding supports the suggestion that perturbations of aromatic amino acid chromophores in proteins due to ligand binding result in red or blue shifts in absorption difference spectra, but in over-all changes of intensity in circular dichroism difference spectra. 相似文献
18.
Spectral studies of cobalt (II)- and Nickel (II)-metallothionein 总被引:1,自引:0,他引:1
The zinc and cadmium of native rabbit metallothionein-1 were replaced stoichiometrically with either cobalt (II) or nickel (II). The electronic, magnetic circular dichroic (MCD), and electron spin resonance spectra of Co (II)-metallothionein reflect distorted tetrahedral coordination of the cobalt atoms. Both the d-d and charge-transfer spectral regions closely resemble those of simple cobalt-tetrathiolate complexes, implying that their coordination chemistry is analogous. Ni (II) complex ions and Ni (II)-metallothionein similarly exhibit analogous MCD bands in the d-d region. The circular dichroic bands associated with ligand-metal charge-transfer transitions in the non-d-d region of Co (II)- and Ni (II)-metallothionein afford additional evidence for the similarity in tetrahedral microsymmetry of the two metal derivatives. The known ratio of 20 thiolate ligands to 7 metal ions, in conjunction with the spectral evidence for tetrathiolate coordination in metallothionein, represents good evidence that these metal thiolates are organized in clusters. 相似文献
19.
20.
The nature of the circular-dichoric spectra of complexes between ribonuclease A and nucleotides.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
S M Dudkin L V Karabashyan M Y Karpeisky S N Mikhailov N S Padyukova V G Sakharovsky 《The Biochemical journal》1977,167(3):749-757
The circular-dichroism and proton-magnetic-resonance spectra of complexes of ribonuclease A with dihydrouridine 3'-phosphate, 2'- and 3'-CMP, arabinosyl-3'-CMP, 1-(2-hydroxyethyl)cytosine 2'-phosphate and 1-(3-hydroxypropyl)cytosine 3'-phosphate were studied. Comparison of the results shows that non-additivity of the circular-dichroic spectrum of an enzyme-nucleotide complex may be due to: (a), alteration of the circular dichroic spectrum of the nucleotide under the influence of the asymmetric protein matrix (induced dichroism), and (b) a change in the nucleotide conformation. The contribution of each of the two factors was estimated to calculate the circular-dichoroic spectra of 2'-CMP and 3'-CMP in complex with ribonuclease A. 3'-CMP in this complex was characterized by negative circular dichroism in the long-wavelength absorption band of the nucleotide, whereas 2'-CMP was characterized by positive circular dichroism. Since both nucleotides in the complex are known to be in an anti conformation, it follows that even small changes in the conformation considerably modify the circular-dichroic spectrum of the nucleotide in complex with the enzyme. 相似文献