Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Folding of bovine pancreatic ribonuclease A (RNase A) is a sequential process which involves the formation of well-populated structural intermediates under suitable conditions. Two intermediates have been detected on the major slow-refolding pathway of RNase A: a late intermediate (IN) which already resembles the native protein in a number of properties and a rapidly formed early intermediate (I1) which shows extensive hydrogen-bonded secondary structure. Here competition experiments between refolding and proteolytic cleavage of the peptide chain are described which yield information about the decrease in accessibility of particular proteolytic cleavage sites during the folding process. Results obtained with pepsin as a proteolytic probe of folding indicate that the primary cleavage site for pepsin, Phe-120-Asp-121, becomes inaccessible early in the course of refolding, if folding is carried out under conditions which effectively stabilize the native state. Under marginally stable conditions, folding is very slow, and protection against peptic cleavage is not detectable prior to the final formation of native protein. The comparison with amide proton exchange experiments suggests that the protection against peptic cleavage occurs during the formation and/or stabilization of hydrogen-bonded secondary structure in the early intermediate (I1). We conclude that the carboxy-terminal region of the RNase peptide chain, which is known to be important for the stability of the folded protein, may also be relevant for early steps of refolding.
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PMID:An early intermediate in the folding of ribonuclease A is protected against cleavage by pepsin. 642 47

A complex of RNase A with a transition-state analog, uridine vanadate, has been studied by a combination of neutron and x-ray diffraction. The vanadium atom occupies the center of a distorted trigonal bipyramid, with the ribose oxygen O2' at the apical position. Contrary to expectations based on the straightforward interpretation of the known in-line mechanism of action of RNase, nitrogen NE2 of histidine-12 was found to form a hydrogen bond to the equatorial oxygen O8, while nitrogen NZ of lysine-41 makes a clear hydrogen bond to the apical oxygen O2'. Nitrogen ND1 of histidine-119 appears to be within a hydrogen-bond distance of the other apical oxygen, O7. Two other hydrogen bonds between the vanadate and the protein are made by nitrogen NE2 of glutamine-11 and by the amide nitrogen of phenylalanine-120. The observed geometry of the complex may necessitate reinterpretation of the mechanism of action of RNase.
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PMID:Active site of RNase: neutron diffraction study of a complex with uridine vanadate, a transition-state analog. 657 1

The aromatic region of the NMR spectrum of bovine pancreatic ribonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37 degrees C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results. Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (2) titratable but similar chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (3) not titratable and different chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues. The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 M NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed.
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PMID:1H nuclear magnetic resonance titration curves and microenvironments of aromatic residues in bovine pancreatic ribonuclease A. 661 20

The 270 MHz 1H NMR spectra of 3'-UMP and 3'-CMP were observed in the presence of a two-fold molar excess of bovine pancreatic RNase A [EC 3.1.27.5] at various pHs. For the C(5), C(6), and C(1') protons of these nucleotides, the pH profiles of chemical shifts induced by binding to RNase A were obtained by plotting the differences between chemical shifts in the presence and the absence of RNase A against pH. Such profiles were bell-shaped for the C(5) and C(6) protons of both 3'-UMP and 3'-CMP. However the profiles of C(1') protons were not bell-shaped but appeared to consist of two bell-shaped curves and reflect the dissociations of at least three ionizable groups. The observations for the C(1') protons suggest that there are at least two forms of complexes different from each other in the interaction reflecting the chemical shift of the C(1') proton. In order to clarify the interacting sites of ribonucleotides affecting the induced shift profile of the C(1') proton, the pH titration curves were observed for 3'-dCMP in the presence of RNase A. The induced shift profile was bell-shaped for the C(1') proton as well as for the C(5) proton of the base. This indicates that the interaction at the O(2')H [or O(2')] sites of ribonucleotides causes the two forms of complexes of 3'-UMP and 3'-CMP with RNase A. The interacting sites and modes were discussed with these and the pH titration curves of His-12, His-119, and Phe-120 of RNase A in the presence of a three-fold molar excess of ribonucleotides.
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PMID:Interaction of substrate analogs with bovine pancreatic ribonuclease A as studied by 1H nuclear magnetic resonance. 665 75

Escherichia coli elongation factor (EF-Tu) binds aminoacyl-tRNAs (aa-tRNA) not only in the presence of GTP but also in the presence of GDP. Complex formation leads to a protection of the aa-tRNA against nonenzymatic deacylation and digestion by pancreatic ribonuclease, as well as to a protection of EF-Tu against proteolysis by trypsin. The equilibrium constant for the binding of Phe-tRNAPheyeast for example to EF-Tu.GDP has been determined to be 0.7 X 10(5) M-1 which is 2 orders of magnitude lower than the equilibrium constant for Phe-tRNAPheyeast binding to EF-Tu.GTP. In the presence of kirromycin, aminoacyl-tRNA binding to EF-Tu.GDP is not affected as much: Phe-tRNAPheyeast is bound with an equilibrium constant of 3 X 10(5) M-1. While there is also a measurable interaction between EF-Tu.GTP and tRNA, such an interaction cannot be detected with EF-Tu.GDP and tRNA, not even at millimolar concentrations. A so far undetected complex formation between aminoacyl-tRNA and EF-Tu.GTP in the presence of pulvomycin, however, could be detected. The results are discussed in terms of the structural requirements of ternary complex formation and in the light of proofreading schemes involving A-site binding on the E. coli ribosome.
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PMID:The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP. 674 37

We have previously demonstrated that (guanine-2-)-methyltransferase activity in extracts from 9,10-dimethyl-1,2-benzanthracene-induced rat mammary tumors differs from that of nonneoplastic mammary tissue. In this report, we explore further the nature of these differences by purification and characterization of the two major transfer RNA (tRNA) (guanine-2-)-methyltransferases from transplantable mammary tumors and proliferating mammary glands from pregnant rats. The position 10-specific (guanine-2-)-methyltransferases (2mGI) from proliferating rat mammary gland and mammary tumor were found to have similar properties with respect to molecular weight, substrate specificity, and elution behavior on ion-exchange columns. In addition, no tissue-specific differences were observed when the mammary tumor and mammary gland 2mGI activities were compared with those of purified rat liver enzyme. In contrast, the position 26-specific (guanine-2-)-methyltransferase (2mGII) from mammary tumors was seen to possess properties different from both the nontumorous mammary gland and liver enzyme. The tumor 2mGII activity showed unusual elution behavior on diethylaminoethyl-Sephadex, eluting along with the 2mGI activity. A small difference in molecular weight was detected between tumor and nontumorous 2mGII activities. Examination of the tumor enzyme in comparison with the well-characterized 2mGII from rat liver indicated that the mammary tumor 2mGII methylated a broader range of tRNA substrates. In particular, mature yeast phenylalanine-specific tRNA, which is methylated in vivo at all major eukaryotic methylation sites and should not be a substrate for eukaryotic methylating enzymes in vitro, could be methylated at low levels by the tumor enzyme. Two-dimensional electrophoretic fingerprint maps of T1 RNase-digested phenylalanine-specific tRNA from Escherichia coli methylated in vitro showed the presence of a methylated oligonucleotide which could not be correlated with normal sites of methylation on the tRNA. From these results, it appears that the mammary tumor 2mGII can methylate at some unusual site(s) on the tRNA molecule.
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PMID:An unusual transfer RNA (guanine-2-)-methyltransferase from transplantable rat mammary tumors. 681 48

The nucleotide sequence of Xenopus laevis phenylalanine tRNA extracted from oocytes was determined to be: pGCCGAAAUAm2GCUCm1AG DDGGGAGAGCm22 G psi psi AGACmUGmAAYA psi C UAAAGm7GDCm5CCUGGT psi CGm1AUCCCGG GUUUCGGCACCAoH. This result was achieved by analysing, with classical procedures [6], the oligonucleotides obtained after digestion by T1 or pancreatic ribonuclease. This sequence is identical to the mammalian sequence. It has been entirely conserved during 10(8) years, the time lapse between the divergence of amphibians and mammals in evolution. In contrast to 5S RNA, no important heterogeneity has been found in the oocyte sequence, suggesting that there is only a single sequence for tRNAphe in X. laevis. Small differences are seen in the elution pattern from RPC-5 columns for immature oocyte and somatic tRNAphe. They are probably due to a submodification of methyl-5-cytidine residues, which appear to be about half methylated in tRNAphe as well as in total tRNA from immature oocytes.
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PMID:The nucleotide sequence of phenylalanine tRNA of Xenopus laevis. 681 27

The degree to which amino acid sequence can be simplified with retention of conformational and functional properties has been investigated by semisynthesis using non-covalent fragment complexes of bovine pancreatic ribonuclease as test cases. Based on the ribonuclease S system, a set of synthetic model sequences was defined for the S-peptide (1-20) region which interacted productively with native S-protein (21-124). The most simple sequence, an eicosapeptide containing helix-favoring Ala residues at all positions except Glu 1 and 14, Phe 8, His 12, and Met 13, effected at least 15% of ribonuclease catalytic activity (versus native ribonuclease S) when added to S-protein in saturating amounts. The data for model S-peptides define an alpha-helical framework and specific side chains at positions 8, 12, and 13 as the core of sequence information necessary for S-peptide to effect a productive non-covalent complex with S-protein. Previous ribonuclease fragment studies also were used as a basis for making the productive, non-overlapping complex, (1-15):(21-111):(116-124). Addition of synthetic (1-15) and (116-124) to (21-111) led to a 3 degrees increase in Tm and 4% (versus ribonuclease A) catalytic activity. The three-fragment complex, with the beta-bend residues 112-115 deleted, exhibited significantly lower stability to thermal denaturation than did related two-fragment complexes. The potential use of three-fragment complexes related to the above is discussed for semi-synthetic sequence modeling concomitantly in the N- and C-terminal regions of ribonuclease.
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PMID:Minimum information content and formation of interacting ribonuclease fragment complexes. 682 84

The enzymically active, semisynthetic, non-covalent complex formed by residues 1 through 118 and residues 111 through 124 of bovine pancreatic ribonuclease A crystallizes at pH 5.2 from (NH4)2SO4/CsCl solution with space group P3(2)21 and unit cell dimensions a and b = 67.7 A, c = 65.1 A and gamma = 120 degrees. The catalytically defective enzyme that results from the replacement of phenylalanine 120 by leucine crystallizes isomorphously with the parent structure (a and b = 67.2 A, c = 64.7 A, gamma = 120 degrees).
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PMID:Crystals of a catalytically defective, semisynthetic ribonuclease isomorphous with those of the fully active parent enzyme. 686 96

Jekowsky et al. reported recently that elongation factor Tu:GTP complex from Escherichia coli protected aminoacyl-tRNA from digestion by pancreatic RNase (I). On the basis of their finding, we have developed the "RNase-resistance assay" for determination of the dissociation constant of aminoacyl-tRNA from aminoacyl-tRNA:EF-Tu:GTP complex. By the use of this sensitive assay, the dissociation constants were estimated to be 3.6 x 10(-7) M for Ala-tRNA1Ala (Torulopsis utilis), 7.9 x 10(-8) M for Phe-tRNAPhe (Escherichia coli), 8.1 x 10(-7) M for initiator Met-tRNAfMet (Escherichia coli), and 5.4 x 10(-6) M for Gly-tRNA1Gly (Staphylococcus epidermidis) participating in cell wall biosynthesis. Moreover, using a relatively large amount of EF-Tu:GTP, we have been able to detect the ternary complexes of initiator Met-tRNAfMet and Gly-tRNA1Gly with EF-Tu:GTP even by the method of gel filtration.
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PMID:Interaction of initiator Met-tRNArMet (Escherichia coli) and Gly-tRNAIGly (Staphylococcus epidermidis) with bacterial elongation factor Tu:GTP complex. 702 61


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