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1.
Y P Venkatesh P J Vithayathil 《International journal of peptide and protein research》1985,25(1):27-32
The influence of chemical mutation featuring the selective conversion of asparagine or glutamine to aspartic or glutamic acid, respectively, on the kinetics of refolding of reduced RNase has been studied. The monodeamidated derivatives of RNase A, viz. RNase Aa1a, Aa1b, and Aa1c having their deamidations in the region 67-74, were found to regain nearly their original enzymatic activity. However, a marked difference in the kinetics of refolding is seen, the order of regain of enzymic activity being RNase A greater than Aa1c congruent to Aa1a greater than Aa1b. The similarities in the distinct elution positions on Amberlite XE-64, gel electrophoretic mobilities, and u.v. spectra of reoxidized and native derivatives indicated that the native structures are formed. The slower rate of reappearance of enzymic activity in the case of the monodeamidated derivatives appears to result from altered interactions in the early stages of refolding. The roles of some amino acid residues of the 67-74 region in the pathway of refolding of RNase A are discussed. 相似文献
2.
B N Manjula A S Acharya P J Vithayathil 《International journal of peptide and protein research》1976,8(3):275-282
A study has been made on the changes in the enzymatic activity of Ribonuclease-A**-(RNase-A) exposed to highly acidic (pH less than 1) acqueous environment. Irreversible alterations of activity were observed when the protein was exposed to an acidic medium for a long period (20 to 60 h). Even prior to these changes in activity RNase-A was found to form intermediates which had very nearly the same activity as the native protein. The primary process in the acid denaturation of RNase-A was observed to be deamidation of the protein leading to the formation of active chromotographically distinct derivatives. The initial product of deamidation, a monodeamidated derivative, has been isolated by chromatography on Amberlite XE-64. This initial deamidation reaction proceeded with very high specificity. The subsequent deamidation reaction is comparatively slower, so that nearly 50% of the native protein could be converted to this derivative before any subsequent deamidation took place. This monodeamidated derivative has been designated RNase-Aa1. The conversion of RNase-A to RNase-Aa1 was not accompanied by any changes in the primary structure other than the observed deamidation. Apart from the differences in chromatographic and electrophoretic mobilities, RNase-Aa1 was found to have very nearly the same activity and physicochemical properties as the native enzyme. Significance of this specific and faster deamidation of RNase-A in this denaturing medium as well as the biological significance of such deamidation reactions of proteins are discussed. 相似文献
3.
Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A. 总被引:3,自引:1,他引:2
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F. Catanzano G. Graziano S. Capasso G. Barone 《Protein science : a publication of the Protein Society》1997,6(8):1682-1693
Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature-induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0-6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 degrees C of denaturation temperature and 65 kJ mol-1 of denaturation enthalpy at pH 5.0. These results are discussed and correlated to the X-ray three-dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine-2'-monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins. 相似文献
4.
At acidic pH, Asp67 and beta-Asp67 (beta-Asp: isoaspartic acid residue) derivatives of RNase A, obtained by selective deamidation of the parent enzyme, spontaneously produces a new derivative containing an aminosuccinyl residue (Asu). The overall secondary structure of the protein chain does not change as a consequence of this substitution, while the catalytic activity on RNA is reduced to about 25%. The pH dependence of the first-order rate constants for the Asu formation has a bell-shaped profile, the maximum being close to the pK(a) of the aspartic acid side chains. Moreover, the values of the rate constants are of the same magnitude of those measured for Asp-containing peptides whose sequence mimics the Asu formation site of the enzyme. This feature indicates that Asp67 and beta-Asp67 residues in the deamidated RNase A derivatives are sited in a region flexible enough to permit the cyclization of the carboxylic side chain to succinimide ring. These results are discussed at the light on to the three-dimensional structure and the thermodynamic stability of the aspartic acid derivatives of RNase A. 相似文献
5.
Kinetic data on the deamidation reaction of Asn67 in RNase A and of Asn3 in the two peptides Ac-Cys-Lys-Asn-Gly-Gln-Thr-Asn-Cys-NH2 and Ac-Cys(Me)-Lys-Asn-Gly-Gln-Thr-Asn-Cys(Me)-NH2, whose sequences are similar to that of the deamidation site in the enzyme, have been determined in a wide range of pH and buffer concentrations. The values of the observed rate constant (k) for the enzyme are markedly lower than those for the peptides. However, the k dependence on pH and buffers is similar for all three substrates, indicating a similar reaction mechanism. The lower k-values for the enzyme have been quantitatively related to the thermal stability and the three-dimensional structure of the enzyme. 相似文献
6.
The initial structural alteration of RNAase A due to acid denaturation (0.5 N HCl, 30 degrees C) that accompanies deamidation (without altering enzymic activity) has been dectected by spectrophotometric titration, fluorescence and ORD/CD measurements. It is shown that acid treated RNAase A has an altered conformation at neutral pH, 25 degrees C. This is characterized by the increased accessibility of buried tyrosine residue(s) towards the solvent. The most altered conformation of RNAase A is found in the 10 h acid-treated derivative. This has about 1.5 additional exposed tyrosine residues and a lesser amount of secondary structure than RNAase A. All three methods (titration, fluorescence and CD) established that the structural transition of RNAase A is biphasic. The first phase occurs within 1 h and the resulting subtle conformational change is constant up to 7 h. Following this, after the release of 0.55 mol of ammonia, the major conformational change begins. The altered conformation of the acid-denatured RNAase A could be reversed completely to the native state through a conformational change induced by substrate analogs like 2'- or 3'-CMP. Thus the monodeamidated derivative isolated from the acid-denatured RNAase A by phosphate is very similar to RNAase A in over-all conformation. The results suggest the possibility of flexibility in the RNAase A molecule that does not affect its catalytic activity, as probed through the tyrosine residues. 相似文献
7.
RNA structure analysis using T2 ribonuclease: detection of pH and metal ion induced conformational changes in yeast tRNAPhe. 总被引:6,自引:6,他引:0
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We describe the use of an enzymic probe of RNA structure, T2 ribonuclease, to detect alterations of RNA conformation induced by changes in Mg2+ ion concentration and pH. T2 RNase is shown to possess single-strand specificity similar to S1 nuclease. In contrast to S1 nuclease, T2 RNase does not require divalent cations for activity. We have used this enzyme to investigate the role of Mg2+ ions in the stabilization of RNA conformation. We find that, at neutral pH, drastic reduction of the available divalent metal ions results in a decrease in the ability of T2 RNase to cleave the anticodon loop of tRNAPhe. This change accompanies an increase in the cleavage of the molecule in the T psi C and in the dihydrouracil loops. Similar treatment of Tetrahymena thermophila 5S ribosomal RNA shows that changes in magnesium ion concentration does not have a pronounced effect on the cleavage pattern produced by T2 RNase. T2 RNase activity has a broader pH range than S1 nuclease and can be used to study pH induced conformational shifts in RNA structure. We find that upon lowering the pH from 7.0 to 4.5, nucleotide D16 in the dihydrouracil loop of tRNAPhe becomes highly sensitive to T2 RNase hydrolysis. This change accompanies a decrease in the relative sensitivity of the anticodon loop to the enzyme. The role of metal ion and proton concentrations in maintenance of the functional conformation of tRNAPhe is discussed. 相似文献
8.
Two distinct regions of angiogenin are critical for angiogenic activity: a catalytic site capable of cleaving RNA and a noncatalytic site, encompassing residues 60-68, which may bind to an endothelial cell-surface receptor [Hallahan, T. W., Shapiro, R., & Vallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2222-2226]. We have now shown that Asn-61 is an essential residue within the cell-binding site and that in addition a segment containing Asn-109 is part of this site. Both asparagines undergo nonenzymatic deamidation during long-term storage or treatment at alkaline pH. While the isolated desamido-61 and desamido-109 derivatives retain nearly full enzymatic activity, their angiogenic activity on the chicken embryo chorioallantoic membrane is markedly attenuated and they do not inhibit angiogenin-induced neovascularization. Tryptic peptide mapping and Edman degradation demonstrate that the isolated deamidated derivatives primarily contain isoaspartic rather than aspartic acid at the positions in question (83% for desamido-61, greater than 99% for desamido-109). Aspartic acid replacement of Asn-61 and Asn-109 by site-directed mutagenesis results in the same ribonucleolytic and angiogenic activities as those of the spontaneous deamidation products. However, the aspartyl derivatives differ strikingly from their isoaspartyl counterparts in that they do inhibit angiogenin-induced angiogenesis. These results indicate that the combination of ribonucleolytic activity and receptor-binding capacity is not sufficient for angiogenic activity and that Asn-61 and Asn-109 within the noncatalytic site are required for some additional function, as yet undefined.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms
(eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used
to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate
conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol,
and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol
was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and
lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation
mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable
tools for screening stabilizers in protein formulations. 相似文献
10.
B. N. Manjula A. Seetharama Acharya Paul J. Vithayathil 《The Biochemical journal》1977,165(2):337-345
Limited proteolysis of RNAase-Aa1 (monodeamidated ribonuclease-A) by subtilisin results in the formation of an active RNAase-S type of derivative, namely RNAase-Aa1S. RNAase-Aa1S was chromatographically distinct from RNAase-S, but exhibited very nearly the same enzymic activity, antigenic conformation and susceptibility to trypsin as did RNAase-S. Fractionation of RNAase-Aa1S by trichloroacetic acid yielded RNAase-Aa1S-protein and RNAase-Aa1S-peptide, both of which are inactive by themselves, but regenerate active RNAase-Aa1S′ when mixed together. RNAase-Aa1S-peptide was identical with RNAase-S-peptide, whereas the protein part was distinct from that of RNAase-S-protein. Titration of RNAase-Aa1S-protein with S-peptide exhibited slight but noticeably weaker binding of the peptide to the deamidated S-protein as compared with that of native protein. Unlike the subtilisin digestion of RNAase-A, which gives nearly 100% conversion into RNAase-S, the digestion of RNAase-Aa1 gives only a 50% conversion. The resistance of RNAase-Aa1 to further subtilisin modification after 50% conversion is apparently due to the interaction of RNAase-Aa1 with its subtilisin-modified product. RNAase-S was also found to undergo activity and structural changes in acidic solutions, similar to those of RNAase-A. The initial reaction product (RNAase-Sa1) isolated by chromatography was not homogeneous. Unlike the acid treatment of RNAase-A, which affected only the S-protein part, the acid treatment of RNAase-S affected both the S-protein and the S-peptide region of the molecule. 相似文献
11.
Selective deamidation of Asn67 of RNase A to beta-Asp67 and Asp67 residues at neutral pH initially produces greater amounts of the beta-Asp derivative. As the reaction proceeds the relative concentration of [Asp67]-RNase A increases and, at equilibrium, becomes predominant. Such a discrepancy between the kinetic and thermodynamic control on reaction products is discussed in light of information from X-ray three-dimensional analysis and the lower thermodynamic stability of the beta-Asp derivative relative to the parent enzyme. 相似文献
12.
Zabrouskov V Han X Welker E Zhai H Lin C van Wijk KJ Scheraga HA McLafferty FW 《Biochemistry》2006,45(3):987-992
Although deamidation at asparagine and glutamine has been found in numerous studies of a variety of proteins, in almost all cases the analytical methodology that was used could detect only a single site of deamidation. For the extensively studied case of reduced bovine ribonuclease A (13,689 Da), only Asn67 deamidation has been demonstrated previously, although one study found three monodeamidated fractions. Here top down tandem mass spectrometry shows that Asn67 deamidation is extensive before Asn71 and Asn94 react; these are more than half deamidated before Asn34 reacts, and its deamidation is extensive before that at Gln74 is initiated. Except for the initial Asn67 site, these large reactivity differences correlate poorly with neighboring amino acid identities and instead indicate residual conformational effects despite the strongly denaturing media that were used; deamidation at Asn67 could enhance that at Asn71, and these enhance that at Gln74. This success in the site-specific quantitation of deamidation in a 14 kDa protein mixture, despite the minimal 1 Da (-NH2 --> -OH) change in the molecular mass, is further evidence of the broad applicability of the top down MS/MS methodology for characterization of protein posttranslational modifications. 相似文献
13.
The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determined. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds. Deamidation of the labile Asn-67 residue of RNase A was followed electrophoretically and chromatographically. At 80 degrees C, similar rates of deamidation were observed for the disulfide-bonded form, which is thermally unfolded, and the reduced form. At 37 degrees C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30-fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn-67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the beta-turn of residues 66-68. 相似文献
14.
Okumura S Saitoh H Wasano N Katayama H Higuchi K Mizuki E Inouye K 《Protein expression and purification》2006,47(1):144-151
A cytotoxic protein Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli was solubilized in 10 mM HCl. Protein concentration of saturated solution of the recombinant Cry45Aa in 10 mM HCl was about 25 times higher than that in the buffer of previous method (in 50 mM sodium carbonate buffer, pH 10.5, containing 1 mM EDTA, and 10 mM dithiothreitol). The Cry45Aa solubilized in the acidic solution was activated by pepsin as an alternative to proteinase K in the previous method. Cytotoxic activity against CACO-2 cells of the pepsin-treated Cry45Aa was almost identical to the proteinase K-treated protein. The pepsin-treated Cry45Aa was purified by cation-exchange chromatography. The concentration of the purified protein was 539 microg/ml, which was 27-fold higher than that of the activated Cry45Aa by the previously method. The cytotoxic activity of the purified protein was stable in broad pH region (pH 2.0-11.0) for 3 days, and 97% cytotoxic activity remained after incubation at 30 degrees C for 360 min. 相似文献
15.
Fleckenstein B Molberg Ø Qiao SW Schmid DG von der Mülbe F Elgstøen K Jung G Sollid LM 《The Journal of biological chemistry》2002,277(37):34109-34116
Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4(+), DQ2-restricted T cells from the celiac lesions. Capillary electrophoresis with fluorescence-labeled gliadin peptides was used to separate and quantify deamidated and transamidated products. In a competition assay, the affinity of TG2 to a set of overlapping gamma-gliadin peptides was measured and compared with their recognition by celiac lesion T cells. Peptides differed considerably in their competition efficiency. Those peptides recognized by intestinal T cell lines showed marked competition indicating them as excellent substrates for TG2. The enzyme fine specificity of TG2 was characterized by synthetic peptide libraries and mass spectrometry. Residues in positions -1, +1, +2, and +3 relative to the targeted glutamine residue influenced the enzyme activity, and proline in position +2 had a particularly positive effect. The characterized sequence specificity of TG2 explained the variation between peptides as TG2 substrates indicating that the enzyme is involved in the selection of gluten T cell epitopes. The enzyme is mainly localized extracellularly in the small intestine where primary amines as substrates for the competing transamidation reaction are present. The deamidation could possibly take place in this compartment as an excess of primary amines did not completely inhibit deamidation of gluten peptides at pH 7.3. However, lowering of the pH decreased the reaction rate of the TG2-catalyzed transamidation, whereas the rate of the deamidation reaction was considerably increased. This suggests that the deamidation of gluten peptides by TG2 more likely takes place in slightly acidic environments. 相似文献
16.
The biochemical properties of the mutant protein D10R/E48R of Escherichia coli RNase HI, in which Asp(10) and Glu(48) are both replaced by Arg, were characterized. This mutant protein has been reported to have metal-independent RNase H activity at acidic pH [Casareno et al. (1995) J. Am. Chem. Soc. 117, 11011-11012]. The far- and near-UV CD spectra of this mutant protein were similar to those of the wild-type protein, suggesting that the protein conformation is not markedly changed by these mutations. Nevertheless, we found that this mutant protein did not show any RNase H activity in vitro. Instead, it showed high-nucleic-acid-binding affinity. Protein footprinting analyses suggest that DNA/RNA hybrid binds to or around the presumed substrate-binding site of the protein. In addition, this mutant protein did not complement the temperature-sensitive growth phenotype of the rnhA mutant strain, E. coli MIC3001, even at pH 6.0, suggesting that it does not show RNase H activity in vivo as well. These results are consistent with a current model for the catalytic mechanism of the enzyme, in which Glu(48) is not responsible for Mg(2+) binding but is involved in the catalytic function. 相似文献
17.
Ntau-Carboxymethylhistidine-12 RNase is active with both RNA and uridine cyclic 2':3'-monophosphate as substrates. Experimental evidence is presented to show that the activity cannot be due to contaminating RNase A, or other RNase-type protein, to the presence of a mixed dimer between 12- and 119-substituted RNases, or to the presence of trace amounts of Ntau-carboxymethylhistidine-12 RNase. The carboxymethyl derivative has approximately 1 and 13 per cent the specific activity of native enzyme against cyclic 2',3'-UMP at pH 5.0 and 8.5, respectively. 相似文献
18.
The single tryptophan residue in ribonuclease T1 [EC 3.1.4.8] was selectively oxidized by ozone to N'-formylkynurenine, which was then converted to kynurenine by acid-catalyzed deformylation in the frozen state. The two enzyme derivatives thus formed, NFK- and Kyn-RNase T1, lost enzymatic activity at pH 7.5, at which native RNase T1 most efficiently catalyzes the hydrolysis of RNA. At pH 4.75, the modified enzymes retained a decreased but distinct enzymatic activity toward RNA without alteration of substrate specificity, and Kyn-RNase T1 was four times more active than NFK-RNase T1. The binding of 3'-GMP to these modified enzymes decreased remarkably at pH 5.5, the optimum pH for binding to the intact enzyme. The gamma-carboxyl group of glutamic acid 58 was still reactive to iodoacetic acid after modification of tryptophan 59. The amounts of the carboxymethyl group introduced into NFK- and Kyn-RNase T1 were 0.36 and 0.59 mol, respectively, under conditions such that quantitative esterification of native RNase T1 takes place. CD spectroscopy indicated that the tertiary structure of the molecule was disordered in NFK-RNase T1, but not significantly in Kyn-RNase T1. It is concluded that tryptophan 59 functions in maintaining the active conformation of the protein structure, particularly in constructing the active environment for a functionally important set of groups involved in the binding of the substrate at the active site, although direct participation of in tryptophan the catalytic function of ribonuclease T1 is unlikely. 相似文献
19.
The common identity of human acidic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) and beta-D-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) as one enzyme and that of acidic beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), beta-D-fucosidase (no allotted EC number) and alpha-L-arabinosidase (alpha-L-arabinofuranoside arabinohydrolase, EC 3.2.1.55) as another enzyme is indicated by similar binding patterns of glycosidase activities of each enzyme to various lectins. by similar ratios between their intra- and extracellular levels in normal and I-cell fibroblasts and by their deficiencies in liver tissues from patients with Gaucher disease and GM1 gangliosidosis, respectively. A third enzyme, neutral beta-D-galactosidase, purified to homogeneity from human liver has been shown to possess all these five glycosidase activities at neutral pH. These neutral enzymic activities were not bound by any of the lectins examined and found to be reduced in liver and spleen of a patient with neutral beta-D-galactosidase deficiency. An additional form of beta-D-xylosidase with optimal activity at pH 7.4 was bound by the fucose-binding lectin from Ulex eurpaeus while no binding was observed for the acidic (pH 4.8) and neutral (pH 7.0) beta-D-xylosidase activities of the multiple glycosidase enzymes. 相似文献
20.
The value of k(cat)/K(M) for catalysis of RNA cleavage by ribonuclease (RNase) A can exceed 10(9) M(-1) s(-1) in a solution of low salt concentration. This value approaches that expected for the diffusional encounter of the enzyme and its substrate. To reveal the physicochemical constraints upon catalysis by RNase A, the effects of salt concentration, pH, solvent isotope, and solvent viscosity on catalysis were determined with synthetic substrates that bind to all of the enzymic subsites and thereby enable a meaningful analysis. The pK(a) values determined from pH-k(cat)/K(M) profiles at 0.010, 0.20, and 1.0 M NaCl are inconsistent with the known macroscopic pK(a) values of RNase A. This incongruity indicates that catalysis of RNA cleavage by RNase A is limited by the rate of substrate association, even at 1.0 M NaCl. The effect of solvent isotope and solvent viscosity on catalysis support this conclusion. The data are consistent with a mechanism in which RNase A associates with RNA in an intermediate complex, which is stabilized by Coulombic interactions, prior to the formation of a Michaelis complex. Thus, RNase A has evolved to become an enzyme limited by physics rather than chemistry, a requisite attribute of a perfect catalyst. 相似文献