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1.
The dynamic structures of two major forms (LD(I) and LD(II) of pig heart lipoamide dehydrogenase, resolved by TEAE-cellulose column chromatography, were studied by fluorescence depolarization. FAD and ANM were used as an intrinsic and an extrinsic fluorescent probe, respectively. In the experiments with bound FAD of lipoamide dehydrogenase, no thermal dependence of the fluorescence depolarization of either enzyme was observed and the values of polarization were close to the theoretical maximum value of 0.5. Both enzymes contained two reactive thiol groups which differed in their reactivities toward ANM. When the enzymes were labeled with one mol of ANM per mol of enzyme, the rotational relaxation times of LD(I) and LD(II) were found to be 18 ns and 196 ns, respectively. These findings indicate that the sement of LD(I) labeled with ANM fluctuates in the order of nanoseconds, whereas this segment of LD(II) is fixed rigidly. On the other hand, when the enzymes were labeled with two mol of ANM per mol of enzyme, both enzymes showed the composite result of fluorescence depolarization due to the motilities of the segment of enzyme and the whole enzyme molecule. These findings indicate that both LD(I) and LD(II) have the non-equivalent motilities of segments containing one reactive thiol group between the two monomers. In other words, the segment containing the ANM binding site of the one monomer is flexible and this segment of the other monomer is fixed rigidly in both enzymes.  相似文献   

2.
The time dependence of the fluorescence of tryptophanyl and flavin residues in lipoamide dehydrogenase has been investigated with single-photon decay spectroscopy. When the two FAD molecules in the enzyme were directly excited the decay could only be analyzed in a sum of two exponentials with equal amplitudes. This phenomenon was observed at 4 degrees C (tau-1 = 0.8 ns, tau-2 = 4.7 ns) and at 20 degrees C (tau-1 = 0.8 ns, tau-2 = 3.4 ns) irrespective of the emission and excitation wavelengths. This result reveals a difference in the nature of the two FAD centers. By excitation at 290 nm the fluorescence decay curves of tryptophan and FAD were obtained. The decays are analyzed in terms of energy transfer from tryptophanyl to flavin residues. The results, which are in good agreement with those obtained previously with static fluorescence methods, show that one of the two tryptophanyl residues within the subunit transfers its excitation energy to the flavin located at a distance of 1.5 nm.  相似文献   

3.
To gain further insight into the difference in substrate specificity between endoglucanase and cellobiohydrolase, the intrinsic fluorescence properties of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from Trichoderma pseudokiningii S-38 were investigated. The results for the spectral characteristics, ligand binding and fluorescence quenching suggest that the fluorescence of two enzymes comes from tryptophan residues, and that tryptophan residue(s) may be involved in the function of the two enzymes. The results also suggest that the binding tryptophan in EG I may be more exposed to solvent than that in CBH I. This interpretation is supported by the observations that the effects of pH upon the fluorescence of EG I are greater than that of CBH I; spectral shifts are different in EG I and CBH I under various conditions, and fluorescence lifetime changes caused by cellobiose binding are larger for EG I than for CBH I.  相似文献   

4.
To gain further insight into the difference in substrate specificity between endoglucanase and cellobiohydrolase, the intrinsic fluorescence properties of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from Trichoderma pseudokiningii S-38 were investigated. The results for the spectral characteristics, ligand binding and fluorescence quenching suggest that the fluorescence of two enzymes comes from tryptophan residues, and that tryptophan residue(s) may be involved in the function of the two enzymes. The results also suggest that the binding tryptophan in EG I may be more exposed to solvent than that in CBH I. This interpretation is supported by the observations that the effects of pH upon the fluorescence of EG I are greater than that of CBH I; spectral shifts are different in EG I and CBH I under various conditions, and fluorescence lifetime changes caused by cellobiose binding are larger for EG I than for CBH I.  相似文献   

5.
Steady state and time-resolved fluorescence studies on native, desulpho and deflavo xanthine oxidase (XO) have been carried out to investigate the conformational changes associated with the replacement of the molybdenum double bonded sulphur by oxygen and the removal of the flavin adenine dinucleotide (FAD). The steady state quenching experiments of the intrinsic tryptophan residues of the enzyme show that all the nine tryptophans are accessible to neutral quencher, acrylamide, in the native as well as desulpho and deflavo enzymes. However, the number of the tryptophan residues accessible to the ionic quenchers, potassium iodide and cesium chloride, increases upon removal of the FAD centre from the enzyme. This indicates that two tryptophan residues move out from the core of the enzyme to the solvent upon the removal of the FAD. The time-resolved fluorescence studies were carried out on the native, desulpho and deflavo XO by means of the time-correlated single photon counting technique, and the data were analysed by discrete exponential and maximum entropy methods. The results show that the fluorescence decay curve fitted best to a three-exponential model with lifetimes tau(1)=0.4, tau(2)=1.4 and tau(3)=3.0 ns for the native and desulpho XO, and tau(1)=0.7, tau(2)=1.7 and tau(3)=4.8 ns for the deflavo XO. The replacement of the molybdenum double bonded sulphur by oxygen in the desulpho enzyme does not cause any significant change of the lifetime components. However, removal of the FAD centre causes a significant change in the shortest and longest lifetime components indicating a conformational change in the deflavo XO possibly in the flavin domain. Decay-associated emission spectra at various emission wavelengths have been used to determine the origin of the lifetimes. The results show that tau(1) and tau(3) of the native and desulpho XO originate from the tryptophan residues which are completely or partially accessible to the solvent but tau(2) corresponds to those residues which are buried in the core of the enzyme and not exposed to the solvent. For deflavo enzyme, tau(2) is red shifted compared to the native enzyme indicating the movement of tryptophan residues from the core of the enzyme to the solvents.  相似文献   

6.
Modification of tryptophan residues in castor bean hemagglutinin (CBH) with N-bromosuccinimide (NBS) was investigated in detail. Tryptophan residues accessible to NBS increased with lowering pH and six tryptophan residues/mol were oxidized at pH 3.0, while two tryptophan residues/mol were oxidized at pH 5.0. From the pH-dependence curve for tryptophan oxidation, we suggest that the extent of modification of tryptophan in CBH is influenced by an ionizable group with pKa = 3.6. The saccharide-binding activity was decreased greatly by modification of tryptophan concomitantly with a loss of fluorescence. A loss of the saccharide-binding activity was found to be principally due to the modification of two tryptophan residues/mol located on the surface of the protein molecule. In the presence of raffinose, two tryptophan residues/mol remained unmodified with retention of fairly high saccharide-binding activity. The results suggest that one tryptophan residue is involved in each saccharide-binding site on each B-chain of CBH.  相似文献   

7.
A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.  相似文献   

8.
Light-induced activation of class II cyclobutane pyrimidine dimer (CPD) photolyases of Arabidopsis thaliana and Oryza sativa has been examined by UV/Vis and pulsed Davies-type electron-nuclear double resonance (ENDOR) spectroscopy, and the results compared with structure-known class I enzymes, CPD photolyase and (6–4) photolyase. By ENDOR spectroscopy, the local environment of the flavin adenine dinucleotide (FAD) cofactor is probed by virtue of proton hyperfine couplings that report on the electron-spin density at the positions of magnetic nuclei. Despite the amino-acid sequence dissimilarity as compared to class I enzymes, the results indicate similar binding motifs for FAD in the class II photolyases. Furthermore, the photoreduction kinetics starting from the FAD cofactor in the fully oxidized redox state, FADox, have been probed by UV/Vis spectroscopy. In Escherichia coli (class I) CPD photolyase, light-induced generation of FADH from FADox, and subsequently FADH? from FADH, proceeds in a step-wise fashion via a chain of tryptophan residues. These tryptophans are well conserved among the sequences and within all known structures of class I photolyases, but completely lacking from the equivalent positions of class II photolyase sequences. Nevertheless, class II photolyases show photoreduction kinetics similar to those of the class I enzymes. We propose that a different, but also effective, electron-transfer cascade is conserved among the class II photolyases. The existence of such electron transfer pathways is supported by the observation that the catalytically active fully reduced flavin state obtained by photoreduction is maintained even under oxidative conditions in all three classes of enzymes studied in this contribution.  相似文献   

9.
Wu X  Takahashi M  Chen SG  Monnier VM 《Biochemistry》2000,39(6):1515-1521
Amadoriases are a novel class of FAD enzymes which catalyze the oxidative deglycation of glycated amino acids to yield corresponding amino acids, glucosone, and H(2)O(2). We previously reported the purification and characterization of two amadoriase isoenzymes from Aspergillus fumigatus and the molecular cloning of amadoriase II. To identify the primary structure of amadoriase I, we prepared a cDNA library from Aspergillus fumigatus and isolated a clone using a probe amplified by polymerase chain reaction with primers designed according to the partial amino acid sequences from peptide mapping. The primary structure of the enzyme deduced from the nucleotide sequence comprises 445 amino acid residues. The enzyme contains 1 mol of FAD as a cofactor, which is covalently linked to Cys342, as determined by mutagenesis analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and electrospray ionization-collisional-activated dissociation tandem mass spectrometry. Sequence alignment studies show that amadoriase I has 22% homology with monomeric sarcosine oxidase in which FAD is also linked to a homologous Cys residue. Amadoriases are of potential importance as tools for uncoupling hyperglycemia and glycation reactions that are thought to play a role in diabetic complications.  相似文献   

10.
Bovine brain glyoxalase I was investigated in order to identify amino acid residues essential for its catalytic activity. This enzyme is a 44-kDa dimeric protein which exhibits a characteristic intrinsic fluorescence, with an emission peak centered at 342 nm. The total of eight tryptophan residues/molecule was estimated by using a fluorescence titration method. Low values of Stern Volmer quenching constants for the quenchers used indicated that the tryptophan residues are relatively buried in the native molecule. Similar results were obtained for glyoxalase I, purified from yeast and human erythrocytes. The activity of bovine brain glyoxalase I was found to be particularly sensitive to 2,3-butanedione and diethylpyrocarbonate, selective reagents for arginine and histidine residues, respectively. A minor effect was observed by treatment of the enzyme with other amino acid-specific reagents. A protective effect of the competitive inhibitor S-hexylglutathione was observed for all reagents used, indicating the presence of modified amino acids in or near the enzyme active site.  相似文献   

11.
The location of tryptophan residues in the actin macromolecule was studied on the basis of the known 3D structure. For every tryptophan residue the polarity and packing density of their microenvironments were evaluated. To estimate the accessibility of the tryptophan residues to the solvent molecules it was proposed to analyze the radial dependence of the packing density of atoms in the macromolecule about the geometric center of the indole rings of the tryptophan residues. The proposed analysis revealed that the microenvironment of tryptophan residues Trp-340 and Trp-356 has a very high density. So these residues can be regarded as internal and inaccessible to solvent molecules. Their microenvironment is mainly formed by non-polar groups of protein. Though the packing density of the Trp-86 microenvironment is lower, this tryptophan residue is apparently also inaccessible to solvent molecules, as it is located in the inner region of macromolecule. Tryptophan residue Trp-79 is external and accessible to the solvent. All residues that can affect tryptophan fluorescence were revealed. It was found that in the close vicinity of tryptophan residues Trp-79 and Trp-86 there are a number of sulfur atoms of cysteine and methionine residues that are known to be effective quenchers of tryptophan fluorescence. The most essential is the location of SG atom of Cys-10 near the NE1 atom of the indole ring of tryptophan residue Trp-86. On the basis of microenvironment analysis of these tryptophan residues and the evaluation of energy transfer between them it was concluded that the contribution of tryptophan residues Trp-79 and Trp-86 must be low. Intrinsic fluorescence of actin must be mainly determined by two other tryptophan residues--Trp-340 and Trp-356. It is possible that the unstrained conformation of tryptophan residue Trp-340 and the existence of aromatic rings of tyrosine and phenylalanine and proline residues in the microenvironments of tryptophan residues Trp-340 and Trp-356 are also essential to their blue fluorescence spectrum.  相似文献   

12.
Chemical modification of tryptophan residues in abrin-a with N-bromosuccinimide (NBS) was studied with regard to saccharide-binding. The number of tryptophan residues available for NBS oxidation increased with lowering pH, and 11 out of the 13 tryptophan residues in abrin-a were eventually modified with NBS at pH 4.0, while 6 tryptophan residues were modified at pH 6.0 in the absence of specific saccharides. Modification of tryptophan residues at pH 6.0 greatly decreased the saccharide-binding ability of abrin-a, and only 2% of the hemagglutinating activity was retained after modification of 3 residues/mol. When the modification was done in the presence of lactose or galactose, 1 out of 3 residues/mol remained unmodified with a retention of a fairly high hemagglutinating activity. However, GalNAc did not show such a protective effect. NBS-oxidation led to a great loss of the fluorescence of abrin-a, and after modification of 3 tryptophan residues/mol, the fluorescence intensity at 345 nm was only 38% of that of the unmodified abrin-a. The binding of lactose to abrin-a altered the environment of the tryptophan residue at the saccharide-binding site of abrin-a, leading to a blue shift of the fluorescence spectrum. The ability to generate such fluorescence spectroscopic changes induced by lactose-binding was retained in the derivative in which 2 tryptophan residues/mol were oxidized in the presence of lactose, but not in the derivative in which 3 tryptophan residues/mol were oxidized in the absence of lactose. Importance of the tryptophan residue(s) in the saccharide-binding of abrin-a is suggested.  相似文献   

13.
The phosphorescence and fluorescence properties of bacterial luciferase (alphabeta) mutants from Xenorhabdus luminescens were investigated. All tryptophans in the alpha and beta subunits were replaced with tyrosines except for one or two tryptophans in the alpha subunit. Because one luciferase mutant (W250) retained only a single tryptophan in the alpha subunit while two other mutants (W182/250 and W194/250) each contained two tryptophans in the alpha subunit, it was possible to deduce the spectral properties of these specific tryptophans (Trp182, Trp194, Trp250). Analyses of the phosphorescence properties were particularly revealing as only a single phosphorescence emission peak at 411-414 nm was observed for the W250 and W194/250 mutants while peaks at 409 and 414 nm could be clearly observed for the W182/250 mutant. Coupled with intrinsic fluorescence quenching experiments, these results show that alphaTrp182 is in a distinctly polar environment while alphaTrp250 is in a hydrophobic region and illustrate the advantages of using phosphorescence to recognize different microenvironments for tryptophan residues.  相似文献   

14.
P K Bandyopadhyay  C W Wu 《Biochemistry》1978,17(19):4078-4085
Nanosecond and steady-state fluorescence spectoscopy were used to probe the environment of the tryptophan residues of Escherichia coli DNA-binding protein. A spectral shift and a change in quantum yield of the protein upon binding to DNA or oligonucleotides indicate that the tryptophan residues are near or at the DNA binding site. The observation of two excited-state lifetimes of the protein indicates that there is heterogeneity in the microenvironments of these tryptophan residues. The "short-lifetime" tryptophan residues are more sensitive to the interaction with DNA than the "long-lifetime" residues. The results of solute-perturbation studies with iodide or acrylamide indicate that there are tryptophan residues near the surface of the protein which are heterogeneous in their accessibility to these quenchers and that they become less accessible after DNA binding. Also, lysine residues of the protein have been shown to be essential to DNA binding by chemical-modification studies. Tyrosine, arginine, and cysteine residues appear not to be involved in this binding process. From studies of the decay of fluorescence anisotropy of the binding protein in the presence and absence of DNA, it has been concluded that (a) the tetrameric binding protein does not dissociate into subuniits upon binding to the oligonucleotide d(pT)16 and (b) the binding protein-fd DNA complex possesses "local flexibility" and, therefore, cannot be described as a continuous, rigid rod.  相似文献   

15.
The solvent accessibilities to the tryptophanyl microenvironments of wild type sperm whale apomyoglobin (apoMb) and two mutants (W7F and W14F) containing a single tryptophan are measured by fluorescence quenching studies. The results are compared to those relative to horse apoMb. In the wild type sperm whale protein, no difference is noticed in the solvent accessibility of the two indole residues, as documented by the values of the Stern-Volmer constants. By contrast, the two tryptophan residues of horse apoMb are exposed to the solvent in a different way, thus indicating that some local conformational differences exist between the two homologous proteins in solution. The single W --> F substitution at either position 7 or 14 determines local conformational changes that increase the accessibility of the remaining indole residue but do not affect the overall architecture of the protein molecule.  相似文献   

16.
Weber J  Senior AE 《Biochemistry》2000,39(18):5287-5294
Catalytic and noncatalytic nucleotide sites of the F(1) sector of ATP synthase were characterized by tryptophan fluorescence techniques. Seven Trp residues inserted in varied microenvironments in the catalytic sites, and one in the noncatalytic sites, were studied in mutant F(1) enzymes which were otherwise devoid of Trp. Parameters measured were fluorescence lifetimes and dynamic and static quenching by acrylamide in the absence or presence of nucleotide. The results indicated that the solution structures of the mutant enzymes were consistent with reported crystal structures. In enzyme with three empty noncatalytic sites, all sites were relatively inaccessible to acrylamide, indicating a closed conformation. In contrast, when the three catalytic sites were empty, they were relatively and equally accessible to acrylamide, indicating an open conformation. This was the case in the presence or absence of Mg(2+). Residue beta-Trp-331 has been extensively used previously to determine nucleotide binding parameters in F(1). Results here showed that in betaY331W mutant F(1), each of the three beta-Trp-331 residues has an unusually long fluorescence lifetime, confirming that each contributes equally to the overall fluorescence signal.  相似文献   

17.
We report the combined use of real-time photo-CIDNP NMR and stopped-flow fluorescence techniques to study the kinetic refolding of a set of mutants of a small globular protein, HPr, in which each of the four phenylalanine residues has in turn been replaced by a tryptophan residue. The results indicate that after refolding is initiated, the protein collapses around at least three, and possibly all four, of the side-chains of these residues, as (i) the observation of transient histidine photo-CIDNP signals during refolding of three of the mutants (F2W, F29W, and F48W) indicates a strong decrease in tryptophan accessibility to the flavin dye; (ii) iodide quenching experiments show that the quenching of the fluorescence of F48W is less efficient for the species formed during the dead-time of the stopped-flow experiment than for the fully native state; and (iii) kinetic fluorescence anisotropy measurements show that the tryptophan side-chain of F48W has lower mobility in the dead-time intermediate state than in both the fully denatured and fully native states. The hydrophobic collapse observed for HPr during the early stages of its folding appears to act primarily to bury hydrophobic residues. This process may be important in preventing the protein from aggregating prior to the acquisition of native-like structure in which hydrophobic residues are exposed in order to play their role in the function of the protein. The phenylalanine residue at position 48 is likely to be of particular interest in this regard as it is involved in the binding to enzymes I and II that mediates the transfer of a phosphoryl group between the two enzymes.  相似文献   

18.
Human apolipoprotein A-II (apo A-II) in solution and associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was investigated by a combination of absorbance and fluorescence methods. Each apo A-II polypeptide chain contains four tyrosine residues but no tryptophan residues. Two and three tyrosine residues, respectively, appear to be buried for apo A-II in aqueous solution and in the lipid-associated protein. The spectroscopic properties of the tyrosine residues of lipid-associated apo A-II were also investigated. Plots of fluorescence intensity against temperature revealed a discontinuity in the region of the phase transition; however, over the same temperature range, there was no change in the exposure of tyrosine residues to the aqueous environment or in their mobility as measured by fluorescence polarization. Near-ultraviolet circular dichroic measurements demonstrated that the environments of the tyrosine residues of lipid-associated apo A-II and nitrated apo A-II were different from that of the apo A-II in solution or in a denatured state. Similar measurements also revealed that the microenvironments around tyrosines of apo A-II bound to DMPC in the gel phase are different from those observed in the liquid crystalline phase. Using environmentally sensitive fluorescence lipid probes, we have previously demonstrated that the polarity of the lipid/water interface of DMPC changes through a phase transition. The observations presented here indicate that these environmental changes also occur at the lipid/protein interface.  相似文献   

19.
In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,2-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme. The sum of the fluorescence intensities of the enzymes was 1.5 x that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme. The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.  相似文献   

20.
色氨酸残基在内切葡聚糖酶分子中的作用   总被引:13,自引:0,他引:13  
内切葡聚糖酶的化学修饰研究表明:色氨酸残基可能位于活性位点,与底物结合有关.荧光光谱测定指出该酶的荧光几乎都来自色氨酸残基,酶分子中色氨酸微环境对pH变化非常敏感,降低pH导致了酶分子构象发生了较大变化,配基结合使酶分子色氨酸微环境产生了改变,引发了与pH诱导不同的构象变化.  相似文献   

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