Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Ribonuclease T1”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed2002

Impact of four (13)C-proline isotope labels on the infrared spectra of ribonuclease T1.

Ribonuclease T1 was biosynthesized, with all four prolines (13)C-labeled in the peptide C[double bond]O bond, using a proline auxotrophic yeast strain of Saccharomyces cerevisiae. The (13)C- and (12)C-proline isotopomers of ribonuclease T1 were investigated by infrared spectroscopy in the thermally unfolded and natively folded state at 80 and 20 degrees C, respectively. In the thermally unfolded state, both proteins established almost indistinguishable spectral features in the secondary structure sensitive amide I region. In contrast, the spectra measured at 20 degrees C revealed substantial qualitative and quantitative differences, though parallel analysis by circular dichroism suggested identical native folds for both isotopomers. Major spectral differences in the infrared spectra were detected at 1626 and 1679 cm(-1), which are diagnostic marker bands for antiparallel beta-sheets in ribonuclease T1 and at 1645 cm(-1), a region that is characteristic for the infrared absorption of irregular structures. Starting with the known three-dimensional structure of ribonuclease T1, the observed effects of the isotope labeling are discussed on the basis of transition dipole coupling between the (12)C[double bond]O and (13)C[double bond]O groups. The experimental results were confirmed by transition dipole coupling calculations of the amide I manifold of the labeled and unlabeled variant.

Aspergillus oryzae↗
PubMed1981

Nuclear magnetic resonance study on the microenvironments of histidine residues of ribonuclease T1 and carboxymethylated ribonuclease T1.

The 270-MHz 1H NMR spectra and fluorescence of ribonuclease T1 and carboxymethylated ribonuclease T1 were measured in aqueous solution. Histidine C4 proton resonances were assigned to individual residues. From the pH dependences of the chemical shifts of histidine C2 and C4 protons, the pKa values of histidine residues were obtained by the non-linear least-squares method. The hydrogen leads to deuterium exchange rates of histidine C2 protons were determined as a measure of the accessibility of histidine residues to the solvent. Each histidine residue of ribonuclease T1 was found to interact with a carboxylate group of an aspartic or glutamic acid residue; in particular, His-40 was shown to interact with Glu-58. Upon carboxymethylation of Glu-58, His-92 and His-27 are more shielded from the solvent while His-40 remains exposed to the solvent. The 67.9-MHz 13C NMR spectra were measured for the 13C-enriched preparation of carboxymethylated ribonuclease T1. From the pH dependence of 13C chemical shift, the pKa value of the carboxymethylated Glu-58 was found to be unusually low, suggesting the formation of an ionic or hydrogen bond between this carboxymethyl group and a positively charged group, possibly of Arg-77.

Chemical Phenomena↗
PubMed1996

Are turns required for the folding of ribonuclease T1?

Ribonuclease T1 (RNase T1) is a small, globular protein of 104 amino acids for which extensive thermodynamic and structural information is known. To assess the specific influence of variations in amino acid sequence on the mechanism for protein folding, circularly permuted variants of RNase T1 were constructed and characterized in terms of catalytic activity and thermodynamic stability. The disulfide bond connecting Cys-2 and Cys-10 was removed by mutation of these residues to alanine (C2, 10A) to avoid potential steric problems imposed by the circular permutations. The original amino-terminus and carboxyl-terminus of the mutant (C2, 10A) were subsequently joined with a tripeptide linker to accommodate a reverse turn and new termini were introduced throughout the primary sequence in regions of solvent-exposed loops at Ser-35 (cp35S1), Asp-49 (cp49D1), Gly-70 (cp70G1), and Ser-96 (cp96S1). These circularly permuted RNase T1 mutants retained 35-100% of the original catalytic activity for the hydrolysis of guanylyl(3'-->5')cytidine, suggesting that the overall tertiary fold of these mutants is very similar to that of wild-type protein. Chemical denaturation curves indicated thermodynamic stabilities at pH 5.0 of 5.7, 2.9, 2.6, and 4.6 kcal/mol for cp35S1, cp49D1, cp70G1, and cp96S1, respectively, compared to a value of 10.1 kcal/mol for wild-type RNase T1 and 6.4 kcal/mol for (C2, 10A) T1. A fifth set of circularly permuted variants was attempted with new termini positioned in a tight beta-turn between Glu-82 and Gln-85. New termini were inserted at Asn-83 (cp83N1), Asn-84 (cp84N1), and Gln-85 (cp85Q1). No detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn may be critical for the proper folding and/or thermodynamic stability of RNase T1.

Amino Acid Sequence↗
PubMed1997

Upstream subsite interactions for oligonucleotide binding with ribonuclease T1.

Ribonuclease T1 (RNase T1) specifically cleaves RNA at guanylyl residues. Previous studies revealed the presence of an enzyme-subsite interaction for adenosine residues of ApGpC and ApGpU substrates (Osterman and Walz (1979) Biochemistry 10, 1984-1988). The binding of ApG and 2'-deoxyadenylyl-(3',5')-guanosine (dApG) with RNase T1 was studied in the pH range 5-9 using ultraviolet difference spectroscopy. The association constants for these dinucleoside monophosphates showed the same pH dependence both of which differed at low pH values with that for the methyl phosphoester of 5'-GMP (MepG). This difference suggested that binding of the adenosine group is strongly dependent on the deprotonation of an enzyme/ligand group with a pKa value of < or = 4.8. delta G zero for ApG binding minus that for MepG at pH > 6 yielded a delta delta G of -1.17 +/- 0.10 kcal/mol which is a measure of the contribution of the adenosine moiety to binding. ApG bound more tightly than dApG with a mean delta delta G value of -0.73 +/- 0.10 kcal/mol which demonstrated the involvement of the adenosine 2'-OH group in binding. These and other comparisons indicated that delta delta G for maximal binding the adenine base per se was -0.44 kcal/mol. delta delta G for binding pdApdG minus that for dApdG (-0.94 kcal/mol) suggested an enzyme subsite for the phosphomonoester group of former ligand.

Adenosine↗
PubMed2003

Determination of the NMR structure of Gln25-ribonuclease T1.

Ribonuclease (RNase) T1 is a guanyloribonuclease, having two isozymes in nature, Gln25- and Lys25-RNase T1. Between these two isozymes, there is no difference in catalytic activity and three-dimensional structure; however, Lys25-RNase T1 is slightly more stable than Gln25-RNase T1. Recently, it has been suggested that the existence of a salt bridge between Lys25 and Asp29/Glu31 in Lys25-RNase T1 contributes to the stability. To elucidate the effects of the replacement of Lys25 with a Gln on the conformation and microenvironments of RNase T1 in detail, the three-dimensional solution structure of Gln25-RNase T1 was determined by simulated-annealing calculations. As a result, the topology of the overall folding was shown to be very similar to that of the Lys25-isozyme except for some differences. In particular, there were two differences in the property of torsion angles of the two disulfide bonds and the conformations of the residues 11-13, 63-66, and 92-93. With regard to the residues 11-13, the lack of the above-mentioned salt bridge in Gln25-RNase T1 was thought to induce the conformational difference of this segment as compared with the Lys25-isozyme. Furthermore, it was proposed that the perturbation of this segment might transfer to the residues 92-93 via the two disulfide bonds.

Binding Sites↗
PubMed1976

The structure and function of ribonuclease T1. XX. Specific inactivation of ribonuclease T1 by reaction with tosylglycolate.

1. Ribonuclease T1 [EC 3.1.4.8] was inactivated by reaction with tosylglycolate (carboxymethyl rho-toluenesulfonate). At pH 5.5 and 8.0, alkylation of the gamma-carboxyl group of glutamic acid-58 appeared to be the predominant reaction and the major cause of inactivation by tosylglycolate, as in the case of the iodoacetate reaction, although the rate of inactivation was slower than that by iodoacetate. At pH 8.0, histidine residues were also alkylated to some extent. 2. The maximal rate of inactivation was observed at around pH 5.5 and the pH dependence of the rate of inactivation suggested the implication of two groups in the reaction, with apparent pKa values of about 3-4 (possibly histidine residue(s)). 3. In the presence of substrate analogs, ribonuclease T1 was markedly protected from inactivation by tosylglycolate at pH 5.5. The extent of protection corresponded to the binding strength of the substrate analog, except for guanosine. Ribonuclease T1 was much less protected from inactivation by guanosine than by 3'-AMP or 3'-CMP, which has a lower binding strength toward ribonuclease T1. This may indicate that glutamic acid-58 is situated in the catalytic site, at which the phosphate moiety of these nucleotides directly interacts. 4. Enzyme which had been extensively inactivated with tosylglycolate at pH 5.5 scarcely reacted with iodoacetate at pH 5.5, suggesting that these reagents react at the same site, i.e. glutamic acid-58. On the other hand, enzyme which had been inactivated almost completely with tosylglycolate at pH 8.0 still reacted with iodoacetate to some extent at pH 8.0, and the modes of reaction of tosylglycolate and iodoacetate toward ribonuclease T1 appeared to be somewhat different.

Adenosine Monophosphate↗
PubMed1977

The structure and function of ribonuclease T1. XXIII. Inactivation of ribonuclease T1 by reversible blocking of amino groups with cis-aconitic anhydride and related dicarboxylic acid anhydrides.

Ribonuclease T1 [EC 3.1.4.8] was inactivated rapidly by treatment at pH 8.0 and 0 degrees C with cis-aconitic anhydride and related dicabroxylic acid anhydrides, including citraconic, maleic, and succinic anhydrides. Under reaction conditions used, roughly 90% inactivation occurred within 30 min. Analyses of the inactivated enzymes indicated that the reaction took place fairly specifically at the alpha-amino group of the N-terminal alanine and the epsilon-amino group of lysine-41. Upon incubation of these inactivated enzymes at pH 3.6 and 37 degreeC, the activity was regenerated to various extents, depending on the nature of the introduced acyl groups. Under these conditions, the enzyme modified with cis-aconitc anhydride or citraconic anhydride recovered much of the origninal activity after 48 h whereas the enzyme modified with maleic anhydride recovered its activity only partially. Practically no activity was regenerated in the case of the enzyme modified with succinic anhydride under these conditions. The inactivation appears to be due mainly to the effect of the carboxyl group introduced at the epsilon-amino group of lysine-41. The results suggest the usefulness of cis-aconitic anhydride as a reversible blocking reagent for amino groups in proteins.

Aconitic Acid↗
PubMed1988

Two-dimensional 1H-NMR investigation of ribonuclease T1. Resonance assignments, secondary and low-resolution tertiary structures of ribonuclease T1.

Ribonuclease T1 was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for the backbone protons of 95 amino acid residues and most of its side-chain protons using sequence-specific assignment procedures. The secondary structure elements of ribonuclease T1 were identified by an investigation of medium- and long-range nuclear Overhauser effects between the backbone and C beta protons. A low-resolution three-dimensional structure of ribonuclease T1 was deduced from qualitative interpretation of long-range nuclear Overhauser effects.

Amino Acid Sequence↗
PubMed1977

The structure and function of ribonuclease T1 XXIV. Preparation and properties of a stable water-insoluble polyacrylamide derivative of ribonuclease T1.

Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme.

Enzymes, Immobilized↗
PubMed1977

Methylated nucleotide sequences in HeLa-cell ribosomal ribonucleic acid. Correlation between the results from 'fingerprinting' hydrolysates obtained by digestion with T1 ribonuclease and with T1 plus pancreatic ribonuclease.

The methylated nucleotide sequences in HeLa-cell rRNA were previously characterized after enzymic digestion of the rRNA by T1 ribonuclease alone or by combined T1 plus pancreatic ribonucleases. For any methylated product occurring in a T1-ribonuclease digest there must be one or more corresponding products in a combined T1-plus-pancreatic-ribonuclease digest. Here we correlate fully the inter-relationship between the methylated products occurring in the two digestion systems. The analysis has led to the resolution of some previous uncertainties and has permitted an almost complete qualitative and quantitative description of the methylated components in HeLa-cell rRNA. The data are compared with those reported by other authors for HeLa-cell rRNA.

Base Sequence↗
PubMed1976

The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide.

1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.

Alanine↗
PubMed1976

The primary structure of non-initiator methionine transfer ribonucleic acid from Bakers' yeast. I. Purification and complete digestion with ribonuclease T1 and pancreatic ribonuclease A.

The methionine acceptor activity of a crude tRNA from bakers' yeast was resolved into two peaks (I and II) by column chromatography on DEAE-Sephadex A-25 with a 1 M phosphate system. Methionine tRNA from peak II was not formylated by E. coli methionyl-tRNA transformylase [EC 2.1.2.9.] after being charged with methionine, whereas that from peak I was formylatable under the same conditions. A substantial amount of unlabelled methionine tRNA, tRNAMetm, was highly purified from the peak II fraction by successive chromatographic procedures. The purified tRNAMetm was digested with pancreatic ribonuclease A [EC 3.1.4.22] and ribonuclease T1 [EC 3.1.4.8]. The digestion products were isolated into individual components and completely sequenced. The results of sequence analysis of the two RNase digests were in good agreement and indicated that the chain length of this tRNA is 76, including 13 modified nucleotides. These oligonucleotide fragments can be constructed into a unique total sequence, assuming a few conventional features of clover leaf structure for the tRNA was established by analyses of partial digestion products with RNase T1, as reported in the accompanying paper.

Base Sequence↗
PubMed1977

The structure and function of ribonuclease T1. XXII. Tryptic cleavages of the single lysyl and arginyl bonds in ribonuclease T1.

1. When ribonuclease T1 [EC 3.1.4.8] was treated with trypsin [EC 3.4.21.4] at pH 7.5 and 37 degrees, activity was lost fairly slowly. At higher temperatures, however, the rate of inactivation was markedly accelerated. The half life of the activity was about 2.5 h at 50 degrees and 1 h at 60 degrees. 3'-GMP and guanosine protected the enzyme significantly from tryptic inactivation. 2. Upon tryptic digestion at 50 degrees, the Lys-Tyr (41-42) and Arg-Val (77-78) bonds were cleaved fairly specifically, yielding two peptide fragments. One was a 36 residue peptide comprizing residues 42 to 77. The other was a 68 residue peptide composed of two peptide chains cross-linked by a disulfide bond between half-cystines -6 and -103, comprizing residues 1 to 41 and 78 to 104. 3. When the trinitrophenylated enzyme, in which the alpha-amino group of alanine-1 and the episolone-amino group of lysine 41 were selectively modified, was treated with trypsin at 37 degrees, the activity was lost fairly rapidly with a half life of about 4 h. In this case, tryptic hydrolysis occurred fairly selectively at the single Arg-Val bond. Thus the enzyme could be inactivated by cleavage of a single peptide bond in the molecule, an indication of the importance of the peptide region involving the single arginine residue at position 77 in the activity of ribonuclease T1.

Amino Acids↗
PubMed1992

Dissection of the ribonuclease T1 subsite. The transesterification kinetics of Asn36Ala and Asn98Ala ribonuclease T1 for minimal dinucleoside phosphates.

Ribonuclease T1 contains a subsite which by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis [Steyaert, J., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8661-8665]. The Asn36Ala and Asn98Ala mutations reduce the transesterification rates of GpA, GpC and GpU considerably whereas they have virtually no effect on the transesterification kinetics of the synthetic substrate guanosine 3'-(methyl phosphate) (GpMe) (in which the leaving nucleoside is replaced by methanol), indicating that the Asn36 and Asn98 side chains are part of the RNase T1 subsite [Steyaert, J., Haikal, A. F., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8666-8670]. The kinetics of the Asn36Ala, Asn98Ala and wild-type catalyzed transesterification of guanosine 3'-(5'-D-ribosyl phosphate) (GpRib), another GpN analog in which the leaving groups is replaced by D-ribose, enables the mapping of the subsite interactions provided by Asn36 and Asn98. We find that the Asn36 amide function contributes 4.6 kJ/mol to catalysis through interactions with the ribose moiety of the leaving nucleoside. Asn98 is at least in part responsible for the subsite preference for cytidine; the Asn98 side chain preferentially binds cytosine as the leaving nucleoside base.

Alanine↗