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1.
The structural flexibility of creatine kinase has been investigated with the covalent hydrophobic probe 2-[4′-(2″-iodoacetamido) phenyl] aminonaphthalene-6-sulfonic acid (IAANS) which reacts at vastly different rates with the two subunits to give a protein conjugate with fluorescence characteristic of reaction with a site in a hydrophobic cleft. Binding of purine nucleotides greatly enhances the probe fluorescence while pyrimidine nucleotides quench the fluorescence. Small anions bind to nucleotide-free creatine kinase near the location of the transferable phosphoryl group and quench both the IAANS fluorescence of modified creatine kinase and the tryptophan fluorescence of native creatine kinase. Chloride and nitrate non-competitively inhibit MgADP binding both with and without creatine. Fluorescence energy transfer demonstrates that the active sites of creatine kinase are well separated and become further apart after the nucleotide-induced conformational change.  相似文献   

2.
The behavior of an array of fluorescent human alpha-thrombin derivatives in reporting binding of the fragment 2 domain of prothrombin was characterized as a representative application of the active-site-selective labeling approach to studies of blood coagulation proteinase regulatory interactions. An array of 16 thrombin derivatives was prepared by affinity labeling of the proteinase active site with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl or N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, followed by selective modification of the NH2OH-generated thiol group on the covalently incorporated inhibitors with each of eight thiol-reactive fluorescence probes. The changes in probe fluorescence intensity of the derivatives, signaling changes in the environment of the catalytic site associated with fragment 2 binding, appeared to be a unique and unpredictable function of the structure of the probe and the connecting peptide. These results demonstrated the utility of the labeling approach for overcoming the problem of not being able to predict which fluorescent label will provide the most useful proteinase derivative for investigating an interaction by enabling a greater variety of them to be prepared and screened for those with the most desirable properties. To determine whether the approach could be extended to other proteinases, the specificity of labeling with the fluorescence probe iodoacetamide, 5-(iodoacetamido)fluorescein, by use of the two thioester inhibitors was evaluated for several other blood coagulation proteinases and related trypsin-like enzymes. All of the proteinases were labeled in an active-site-selective manner. The combined results of quantitating the labeling reactions for the proteinase and inhibitor combinations studied thus far showed active-site-specific incorporation of 0.98 +/- 0.10 mol of inhibitor/mol of active sites and 0.92 +/- 0.11 mol of probe/mol of active sites, representing an overall greater than or equal to 93% site-specificity of labeling. These results demonstrated the broad applicability of the labeling approach for fluorescence studies of proteinases that differ greatly in their catalytic specificities.  相似文献   

3.
A new polarity-sensitive fluorescent probe, 3-(4-chloro-6-p-glyoxal-phenoxy-1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CGTDP), is synthesized for selective labeling of active-site arginine residues. The probe comprises a neutral red moiety as a polarity-sensitive fluorophore and a phenylglyoxal unit as an arginine-specific labeling group. The probe exhibits a sensitive response of shift of fluorescence maximum emission wavelength to solvent polarity only instead of pH or temperature, which leads to the use of the probe in detecting the local polarity and conformational changes of the active site of rabbit muscle creatine kinase (CK) denatured by pH or temperature. The polarity of the active site domain has been first found to correspond to a dielectric constant of about 44, and the conformational change of the active site directly revealed by CGTDP occurs far before that of CK as a whole disclosed by the intrinsic tryptophan fluorescence during acid or thermal denaturation. The present strategy may provide a useful method to detect the local polarity and conformational changes of the active sites of many enzymes that employ arginine residues as anion recognition sites under different denaturation conditions.  相似文献   

4.
S. lignicolum acid proteinases A-1, A-2, and B (S-PI-insensitive) were examined for the structure of the active sites by the method of Nakatani using PAD as a probe. We attempted to screen for substances that release zinc(II)-PAD from the complex of zinc(II)-PAD-S-PI-insensitive acid proteinases. Angiotensin I released zinc(II)-PAD from the complex as well as from the complex of S-PI-sensitive acid proteinases. Angiotensin I is a good substrate for these enzymes regardless of S-PI-sensitivity. These results suggest that the two carboxyl groups capable of binding to zinc(II)-PAD are at the active site regions of S. lignicolum enzymes and that they are involved in their catalytic actions.  相似文献   

5.
The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.  相似文献   

6.
Based on the concept of sequence conservation around the active sites of serine proteinases, polymerase chain reaction applied to mRNA amplification allowed us to obtain a 260-bp probe which was used to screen a mouse pituitary cDNA library. The primers used derived from the cDNA sequence of active sites Ser* and Asn* of human furin. Two cDNA sequences were obtained from a number of positive clones. These code for two similar but distinct structures (mPC1 and mPC2), each being homologous to yeast Kex2 and human furin. In situ hybridization (mPC1) and Northern blots (mPC1 = 3.0 kb and mPC2 = 2.8 and 4.8 kb) demonstrated tissue and cellular specificity of expression, only within endocrine and neuroendocrine cells. These data suggest that mPC1 and mPC2 represent prime candidates for tissue-specific pro-hormone converting proteinases.  相似文献   

7.
To obtain the local information on the tryptophan domain in a protein, the design and synthesis of a new fluorescent probe, 1,7-bis(4-hydroxy-3-methoxyphenyl)-4-diazo-1,6-heptadiene-3,5-dione, is reported for the selective modification of tryptophan residues. The probe comprises a curcumin fluorophore and a diazo labeling group, whose spectroscopic properties are characterized. The diazo group may be catalytically degraded by transition metal complexes such as Rh2(OAc)4, generating an active rhodium carbenoid intermediate, which can react selectively with tryptophan residues. By the use of the carbene's intermolecular reactions, the tryptophan residue (Trp19) of beta-lactoglobulin may be modified with the diazo curcumin probe. Furthermore, slight secondary but larger tertiary structural changes are detected after Trp19 is modified, and the Trp19 modification produces a great effect on the binding of 8-anilino-1-naphthalenesulfonic acid and retinol. These results indicate that the Trp19 residue plays an essential role in the structure and stability of beta-lactoglobulin, and the specific modification of this residue may have a potential use in further elucidating the relationship between the structure and function of the protein.  相似文献   

8.
The trp RNA-binding attenuation protein (TRAP) is a paradigmatic allosteric protein that regulates the tryptophan biosynthetic genes associated with the trp operon in bacilli. The ring-shaped 11-mer TRAP is activated for recognition of a specific trp-mRNA target by binding up to 11 tryptophan molecules. To characterize the mechanisms of tryptophan-induced TRAP activation, we have performed methyl relaxation dispersion (MRD) nuclear magnetic resonance (NMR) experiments that probe the time-dependent structure of TRAP in the microsecond-to-millisecond "chemical exchange" time window. We find significant side chain flexibility localized to the RNA and tryptophan binding sites of the apo protein and that these dynamics are dramatically reduced upon ligand binding. Analysis of the MRD NMR data provides insights into the structural nature of transiently populated conformations sampled in solution by apo TRAP. The MRD data are inconsistent with global two-state exchange, indicating that conformational sampling in apo TRAP is asynchronous. These findings imply a temporally heterogeneous population of structures that are incompatible with RNA binding and substantiate the study of TRAP as a paradigm for probing and understanding essential dynamics in allosteric, regulatory proteins.  相似文献   

9.
Spin-labeled fluorophosphonates react with serine proteinases and esterases in a manner analogous to the common active phosphate ester inhibitors. The reporter group properties of the spin-labeled inhibitors enable a comparative study of the micro structure of the active sites of these enzymes as well as a facile determination of rates of inhibition. The effect of various environmental perturbations on the conformation of these active sites has also been probed.  相似文献   

10.
11.
The spin-labeled tryptophan was used as a structural probe of hemoglobin contact sites. The ESR spectral data indicated that the probe exhibits weak binding to hemoglobin with a dissociation constant of 3.2.10(-5) and 4.0 mol bound per hemoglobin tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.2 ns. The topology of the contact sites was investigated by using dual spin-label methodology in which spin-labeled tryptophan and (2H,15N) substituted and deuterated maleimide spin label [2H-15N]MSL covalently-bound to Cys-beta 93 residue were used. The ESR spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled hemoglobin. The environment of the contact sites is discussed.  相似文献   

12.
In a new strategy for labeling the active sites of serine proteinases with fluorescence probes (Bock, P. E. (1988) Biochemistry 27, 6633-6639), a thioester peptide chloromethyl ketone inhibitor is incorporated into the enzyme active center and used to produce a unique thiol group which provides a site for selective chemical modification with any one of many thiol-reactive fluorescence probes. This approach was developed to increase the opportunities for identifying fluorescent proteinase derivatives that act as reporters of binding interactions by allowing a large number of derivatives, representing a broad range of probe spectral properties, to be readily prepared. In the studies described here, the specificity of the labeling approach was evaluated quantitatively for the labeling of human alpha and beta/gamma-thrombin with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl and N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, and the thiol-reactive fluorescence probe, 5-(iodoacetamido)fluorescein. Irreversible inactivation of thrombin by the inhibitors was accompanied by incorporation of 0.98 +/- 0.06 mol/mol of the thioester group into the active site, independent of a 470-fold difference between the thioester peptide chloromethyl ketones in the bimolecular rate constants of alpha-thrombin affinity labeling. Subsequent mild treatment of the covalent thrombin-inhibitor complexes with NH2OH in the presence of 5-(iodoacetamido)fluorescein resulted in generation of the thiol group together with its selective modification and incorporation of 0.96 +/- 0.07 mol of probe/mol of active sites. The incorporated label was localized to a 9000 molecular weight region of alpha and beta/gamma-thrombin containing the catalytic-site histidine residue. Evaluation of competing, side reactions showed that they did not significantly compromise the active site specificity of labeling. These results demonstrated equivalent, active-site-selective fluorescence probe labeling of alpha and beta/gamma-thrombin by use of either of the thioester peptide chloromethyl ketones, with a site specificity of greater than or equal to 94%.  相似文献   

13.
The spin-labeled tryptophan (TrpSL) was used as a structural probe of hemoglobin (Hb) contact sites. The electron paramagnetic resonance spectral data indicated that the probe exhibits weak binding to Hb with a dissociation constant of 3.2 x 10(-5) and 4.0 mol bound per Hb tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.5 s. The topology of the contact sites was investigated by using a dual spin label methodology in which TrpSL and 2H-15N covalently bound to B 93 cysteine residue were used. The electron spin resonance spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled Hb. The environment of the contact sites is discussed.  相似文献   

14.
The spin-labeled tryptophan was used as a structural probe of hemoglobin contact sites. The ESR spectral data indicated that the probe exhibits weak binding to hemoglobin with a dissociation constant of 3.2 · 10−5 and 4.0 mol bound per hemoglobin tetramer. The spectrum suggested that the bound tryptophan was ‘partially immobilized’ with a correlation time reflecting the environment of the tryptophan binding site of 8.2 ns. The topology of the contact sites was investigated by using dual spin-label methodology in which spin-labeled tryptophan and (2H,15N) substituted and deuterated maleimide spin label [2H-15N]MSL covalently-bound to Cys-ß93 residue were used. The ESR spectral data suggested that the tryptophan binding sites were located within 8–10Åof the nitroxide free radical of spin-labeled hemoglobin. The environment of the contact sites is discussed.  相似文献   

15.
The calpains form a growing family of structurally related intracellular multidomain cysteine proteinases containing a papain-related catalytic domain, whose activity depends on calcium. The calpains are believed to play important roles in cytoskelatel remodeling processes, cell differentiation, apoptosis and signal transduction, but are also implicated in a number of diseases. Recent crystal structures of truncated rat and full-length human apo-m-calpain revealed the domain arrangement and explained the inactivity of m-calpain in the absence of calcium by a disrupted catalytic domain. Proteolysis studies have indicated several susceptible sites, in particular in the catalytic subdomain IIb and in the following domain III, which are more accessible to attacking proteinases in the presence than in the absence of calcium. The current view is that m-calpain exhibits a number of calcium binding sites, which upon calcium binding cooperatively interact, triggering the reformation of a papain-like catalytic domain, accompanied by enhanced mobilisation of the whole structure. To further analyse the flexibility of m-calpain, we have determined and refined the human full-length apo-m-calpain structure of a second crystal form to 3.15 A resolution. Here we present this new structure, compare it with our first structure now re-refined with tighter constrain parameters, discuss the flexibility in context with the proteolysis and calcium binding data available, and suggest implications for the calcium-induced activation process.  相似文献   

16.
Photochemically induced dynamic nuclear polarization was used to study the accessibility of surface tyrosine and tryptophan residues in proteinases, in their protein inhibitors and in the proteinase–inhibitor complexes. The accessibility probe is the triplet of 10-(carboxyethyl) flavin formed by optical excitation. On complex formation we observe accessibility loss in the surface tyrosines and tryptophans in the proximity of the proteinase–inhibitor contact site, and in the case of bovine pancreatic trypsin inhibitor, in more distant tyrosines as well.  相似文献   

17.
A fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases is described. It consists of: selective labelling of the corresponding subsites with a fluorescent group by using N alpha-dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ated peptide chloromethanes containing different numbers of amino acid residues, and probing the immediate environment of the subsites by quenching experiments using ionic and neutral quenchers. Intramolecular distances between the subsites and particular chromophores can be also determined. The technique is of general applicability to all serine proteinases. The above mentioned approach was applied to two proteinases: subtilisin Novo and mesentericopeptidase. It was concluded that the substrate-binding site of mesentericopeptidase is considerably more polar than that of subtilisin Novo. Intramolecular distances between the labelled subsites and tryptophan residues in the two proteinases were determined.  相似文献   

18.
Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.  相似文献   

19.
Some structural properties of Neurospora tyrosinase have been studied by fluorescence spectroscopy. The emission spectra observed for oxy-, deoxy-, met- and apo-tyrosinase and the Co2+-substituted form are indicative of a protein containing buried tryptophan residues. By using acrylamide and iodide, part of the emission is quenched, indicating heterogeneity in the tryptophan environment. Upon binding of Cu2+ or Co2+ to apo-tyrosinase, a marked decrease of the tryptophan quantum yield is observed. A further decrease in emission intensity results from the binding of molecular O2 to the deoxy form. The fluorescent probe 8-anilinonaphthalene-1-sulphonate binds to tyrosinase only when the metal ions are removed. Reconstitution of apo-tyrosinase with Cu2+ completely displaces the probe, suggesting that 8-anilinonaphthalene-1-sulphonate binds to apo-tyrosinase at the active site. The fluorescence properties of Neurospora tyrosinase are compared with those of haemocyanin.  相似文献   

20.
In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

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