Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mechanisms for the ribonuclease T1 (RNase T1; EC 3.1.27.3) catalyzed transesterification reaction generally include the proposal that Glu58 and His92 provide general base and general acid assistance, respectively [Heinemann, U., & Saenger, W. (1982) Nature (London) 299, 27-31]. This view was recently challenged by the observation that mutants substituted at position 58 retain high residual activity; a revised mechanism was proposed in which His40, and not Glu58, is engaged in catalysis as general base [Nishikawa, S., Morioka, H., Kim, H., Fuchimura, K., Tanaka, T., Uesugi, S., Hakoshima, T., Tomita, K., Ohtsuka, E., & Ikehara, M. (1987) Biochemistry 26, 8620-8624]. To clarify the functional roles of His40, Glu58, and His92, we analyzed the consequences of several amino acid substitutions (His40Ala, His40Lys, His40Asp, Glu58Ala, Glu58Gln, and His92Gln) on the kinetics of GpC transesterification. The dominant effect of all mutations is on Kcat, implicating His40, Glu58, and His92 in catalysis rather than in substrate binding. Plots of log (Kcat/Km) vs pH for wild-type, His40Lys, and Glu58Ala RNase T1, together with the NMR-determined pKa values of the histidines of these enzymes, strongly support the view that Glu58-His92 acts as the base-acid couple. The curves also show that His40 is required in its protonated form for optimal activity of wild-type enzyme. We propose that the charged His40 participates in electrostatic stabilization of the transition state; the magnitude of the catalytic defect (a factor of 2000) from the His40 to Ala replacement suggests that electrostatic catalysis contributes considerably to the overall rate acceleration. For Glu58Ala RNase T1, the pH dependence of the catalytic parameters suggests an altered mechanism in which His40 and His92 act as base and acid catalyst, respectively. The ability of His40 to adopt the function of general base must account for the significant activity remaining in Glu58-mutated enzymes.
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PMID:Histidine-40 of ribonuclease T1 acts as base catalyst when the true catalytic base, glutamic acid-58, is replaced by alanine. 198 Feb 11

The ribonuclease T1 (RNase T1) gene was ligated to a synthetic gene for the signal peptide of Escherichia coli alkaline phosphatase. When this fusion gene was expressed in E. coli under the control of the trp promoter, active RNase T1 having the correct N-terminal sequence was secreted into the periplasmic space, indicating that the heterologous signal peptide had been cleaved off correctly. The enzyme could be readily purified from the periplasmic fraction with a yield of 1.8 mg from 1 liter culture. Adopting the same strategy, it was possible to produce a labile mutant of RNase T1 (Glu-58----Ala mutant) in E. coli, the yield of the purified mutant enzyme being 2.0 mg from 1 liter culture.
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PMID:Secretion of recombinant ribonuclease T1 into the periplasmic space of Escherichia coli with the aid of the signal peptide of alkaline phosphatase. 211 13

Coulombic interactions between charges on the surface of proteins contribute to stability. It is difficult, however, to estimate their importance by protein engineering methods because mutation of one residue in an ion pair alters the energetics of many interactions in addition to the coulombic energy between the two components. We have estimated the interaction energy between two charged residues, Asp-12 and Arg-16, in an alpha-helix on the surface of a barnase mutant by invoking a double-mutant cycle involving wild-type enzyme (Asp-12, Thr-16), the single mutants Thr----Arg-16 and Asp----Ala-12, and the double mutant Asp----Ala-12, Thr----Arg-16. The changes in free energy of unfolding of the single mutants are not additive because of the coulombic interaction energy. Additivity is restored at high concentrations of salt that shield electrostatic interactions. The geometry of the ion pair in the mutant was assumed to be the same as that in the highly homologous ribonuclease from Bacillus intermedius, binase, which has Asp-12 and Arg-16 in the native enzyme. The ion pair does not form a hydrogen-bonded salt bridge, but the charges are separated by 5-6 A. The mutant barnase containing the ion pair Asp-12/Arg-16 is more stable than wild type by 0.5 kcal/mol, but only a part of the increased stability is attributable to the electrostatic interaction. We present a formal analysis of how double-mutant cycles can be used to measure the energetics of pairwise interactions.
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PMID:Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles. 224 51

Ribonuclease T1 (RNase T1) and mutants Gln25----Lys, Glu58----Ala, and the double mutant were prepared from a chemically synthesized gene, cloned and expressed in Escherichia coli. The wild-type RNase T1 prepared from the cloned gene was identical in every functional and physical property examined to RNase T1 prepared from Aspergillus oryzae. Urea and thermal unfolding experiments show that Gln25----Lys is 0.9 kcal/mol more stable and Glu58----Ala is 0.8 kcal/mol less stable than wild-type RNase T1. In the double mutant, these contributions cancel and the stability does not differ significantly from that of wild-type RNase T1. For the double mutant, the dependence of delta G on urea concentration is significantly greater than for wild-type RNase T1 or the single mutants. This suggests that the double mutant unfolds more completely in urea than the other proteins. The activity of Gln25----Lys is identical with that of wild-type RNase T1. The activities of Glu58----Ala and the double mutant are 7% of wild-type when GpC hydrolysis is measured (due to a 35-fold decrease in kcat), and 37% of wild-type when RNA hydrolysis is measured. Thus, Glu58 is important, but not essential to the activity of RNase T1.
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PMID:Conformational stability and activity of ribonuclease T1 and mutants. Gln25----Lys, Glu58----Ala, and the double mutant. 266 37

The genes for ribonuclease T1 and its site-specific mutants were chemically synthesized and introduced to Escherichia coli. All enzymes were fusion products produced by joining the synthetic gene at specific restriction sites to the synthetic gene for human growth hormone in a plasmid containing the E. coli trp promoter. The fusion protein from this plasmid contained 66% of the amino-terminal sequences of the human growth hormone, which were recognizable immunologically. RNase T1 or its mutants were cleaved from the fusion protein with cyanogen bromide. The synthetic RNase T1 endowed with the revised wild-type triad Gly-Ser-Pro, residues 71-73, was fully functional, readily hydrolyzing pGpC bonds, whereas a mutant enzyme having the originally reported, erroneous triad Pro-Gly-Ser was totally inactive. Various amino acid substitutions were also introduced to the guanosine recognition region comprised of residues 42-45, Tyr-Asn-Asn-Tyr. Substitution of either of the tyrosine residues noted above with phenylalanine had no dramatic effect on the enzyme's function. Replacement of asparagine-43 with arginine or alanine also caused only a small change in the hydrolyzing activity--a mutant enzyme maintained greater than 50% of the wild-type activity. In sharp contrast, when aspartic acid or alanine was substituted for asparagine-44, the activity was dramatically reduced to a few percent of the wild-type activity.
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PMID:Inquiries into the structure-function relationship of ribonuclease T1 using chemically synthesized coding sequences. 301 4

The Glu 46 of ribonuclease T1, which is assumed to interact with Nl of the guanine residue in RNA by a hydrogen bond from the result of X-ray analysis, was changed to alanine by site-directed mutagenesis and its function examined. The nucleolytic activity of the Ala 46 mutant enzyme against pGpC decreased to 0.4% of that of the wild-type enzyme, on the other hand its activity against pApC increased. This result suggests that the Glu 46 is essential for the recognition of the guanine base but that it also interferes with the recognition of the adenine base.
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PMID:Glu 46 of ribonuclease T1 is an essential residue for the recognition of guanine base. 312 58

Ribonuclease T1 (RNase T1, EC 3.1.27.3) is a guanosine-specific ribonuclease that cleaves the 3',5'-phosphodiester linkage of single-stranded RNA. It is assumed that the reaction is generated by concerted acid-base catalysis between residues Glu-58 and His-92 or His-40. From the results of chemical modification and NMR studies, it appeared that the residue Glu-58 was indispensable for nucleolytic activity. However, we have recently demonstrated that Glu-58 is an important but not an essential residue for catalytic activity, using the methods of genetic engineering to change Glu-58 to Gln-58 etc [Nishikawa, S., Morioka, H., Fuchimura, K., Tanaka, T., Uesugi, S., Ohtsuka, E., & Ikehara, M. (1986) Biochem. Biophys. Res. Commun. 138, 789-794]. In the present paper, we report that mutants of RNase T1 with residue Ala-40 or Ala-92 have almost no activity, while mutants that contain Ala-58 retain considerable activity. These results show that the two histidine residues, His-40 and His-92, but not Glu-58, are indispensable for the catalytic activity of the enzyme. We propose a revised reaction mechanism in which two histidine residues play a major role, as they do in the case of RNase A.
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PMID:Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A. 312 7

Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.
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PMID:Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering. 315 Oct 17

1. RNase Ms, a base non-specific RNase from Aspergillus saitoi was reduced and carboxymethylated (RCM-RNase Ms). RCM-RNase Ms was hydrolyzed with trypsin, and the trypsin digests were then treated with chymotrypsin. Trypsin digests were also treated with Staphylococcus protease and with chymotrypsin, separately. 2. By the analyses of the amino acid sequences of the peptides formed, the alignment of these peptides in RCM-RNase Ms was determined. 3. From the digest of heat-denatured RNase Ms with Bacillus subtilis protease, two peptides containing disulfide bridges were isolated. From the analysis of these two peptides, the locations of the bridges were determined. 4. The amino acid sequence of RNase Ms was compared with those of RNase T1 (Asp. oryzae, guanine specific), RNase U1 (Ustilago sphaerogena, guanine specific) and RNase U2 (Ustilago sphaerogena, purine specific). There are very similar sequences between these for RNases irrespective of their differences in base specificity. These were, in RNase Ms, tripeptide sequence containing His39 (Tyr-Pro-His), the tetrapeptide containing Glu57 (Glu-Tyr-Pro-Ile), the hexapeptide containing Arg76 (Asp-Arg-Val-Ile-Phe-Asp) and the hexapeptide containing His 91 (Ile-Thr-His-Thr-Gly-Ala). The other sequences common for all four RNases are Tyr67, Phe100, and Cys103 in RNase Ms. Since among these peptides His39, Glu57, His91, and Arg76 in RNase Ms corresponded to His40, Glu58, His92, and Arg77 in RNase T1 which are known to be involved in the active site of RNase T1, the possible role of these amino acids in the active site of RNase Ms is discussed. 5. The sequence similarity of RNase Ms to that of RNase T1 was about 60% and to those of RNase U1 and RNase U2 was about 30%. 6. The details of the experimental evidence used to elucidate the amino acid sequence of RNase Ms are described in the supplemental miniprint.
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PMID:Primary structure of a minor ribonuclease from Aspergillus saitoi. 709 2

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that catalyse protein folding both in vitro and in vivo. We isolated a peptidyl-prolyl cis/trans isomerase (PPIase) which is specifically associated with the 50S subunit of the Escherichia coli ribosome. This association was abolished by adding at least 1.5 M LiCl. Sequencing the N-terminal amino acids in addition to three proteolytic fragments totalling 62 amino acids revealed that this PPIase is identical to the E.coli trigger factor. A comparison of the amino acid sequence of trigger factor with those of other PPIase families shows little similarities, suggesting that trigger factor may represent an additional family of PPIases. Trigger factor was purified to homogeneity on a preparative scale from E.coli and its enzymatic properties were studied. In its activity towards oligopeptide substrates, the trigger factor resembles the FK506-binding proteins (FKBPs). Additionally, the pattern of subsite specificities with respect to the amino acid preceding proline in Suc-Ala-Xaa-Pro-Phe-4-nitroanilides is reminiscent of FKBPs. However, the PPIase activity of the trigger factor was not inhibited by either FK506 or by cyclosporin A at concentrations up to 100 microM. In vitro, the trigger factor catalysed the proline-limited refolding of a variant of RNase T1 much better than all other PPIases that have been examined so far.
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PMID:A ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor. 758 23


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