Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The energetics of complementary packing of nonpolar side chains in the hydrophobic core of a protein were analyzed by protein engineering experiments. We have made the mutations Ile----Val, Ile----Ala, and Leu----Ala in a region of the small bacterial ribonuclease barnase where the major alpha-helix packs onto the central beta-sheet. The destabilization resulting from the creation of cavities was determined by measuring the decrease in free energy of folding from reversible denaturation induced by urea, guanidinium chloride, or heat. The different methods give consistent and reproducible results. The loss in free energy of folding for the mutant proteins is 1.0-1.6 kcal/mol per methylene group removed. This exceeds by severalfold the values obtained from model experiments of the partitioning of relevant side chains between aqueous and nonpolar solvents. Much of this discrepancy arises because two surfaces are buried when a protein folds--both the amino acid side chain in question and the portions of the protein into which it packs. These experiments directly demonstrate that the interior packing of a protein is crucial in stabilizing its three-dimensional structure: the conversion of leucine or isoleucine to alanine in the hydrophobic core loses half the net free energy of folding of barnase with a concomitant decrease in yield of the expressed recombinant protein.
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PMID:Energetics of complementary side-chain packing in a protein hydrophobic core. 266 64

The urinary enzymes alanine amino-peptidase, alkaline phosphatase, gamma-glutamyltransferase and N-acetyl-beta-D-glucosaminidase and the two urine low-molecular mass proteins lysozyme and ribonuclease were measured in 30 healthy men and 36 insulin-dependent diabetics. 17 diabetics had "clinical proteinuria" (greater than 7.5 g/mol creatinine) and were defined as patients with manifest diabetic nephropathy. The remaining 19 diabetics were without proteinuria. The excretion rates of the two urine proteins and all enzymes except for gamma-glutamyltransferase were the highest in patients suffering from diabetic nephropathy. The excretion rates in both diabetic groups exceeded those of the control group. N-Acetyl-beta-D-glucosaminidase was more often increased than albumin in diabetics without manifest diabetic nephropathy. It is concluded that the tubular dysfunction is an early indicator of the incipient diabetic nephropathy. Thus, tubular parameters, especially the lysosomal enzyme N-acetyl-beta-D-glucosaminidase may be used in follow-up studies of diabetics.
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PMID:[Urine enzymes and low molecular weight proteins as indicators of diabetic nephropathy]. 273 55

The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Amino acid sequence of human tumor derived angiogenin. 286 94

Ribonuclease A was studied by two-dimensional 1H NMR spectroscopy. 10 out of 12 alanine and 9 out of 10 threonine spin systems as well as all valine [9], leucine [2] and isoleucine [3] spin systems were identified from the correlated spectroscopy (COSY) and relayed coherence transfer spectroscopy (RCT). Sequence-specific assignments were obtained from nuclear Overhauser effect spectra for proton resonances of 21 amino acid moieties. 2' and 3'-pyrimidine-nucleotide-RNase-A complexes were also investigated by two-dimensional NMR. We were able to monitor structural changes in the active center, the vicinity of the active center and in regions far from the catalytic region. Chemical shift changes of resonances of protons near Thr-45 reflected the binding of the same moiety. This in turn is also dependent on the position of the nucleotide phosphate group. Binding of 2' nucleotides led to characteristic changes in protein regions not affected by the binding of 3' nucleotides. These results are interpreted in terms of structural differences between the 2' and 3'-nucleotide-RNase-A complexes; the structure of the complex of the native 3' nucleotide inhibitor being more closely related to that of the free protein.
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PMID:Two-dimensional 1H NMR investigation of ribonuclease A and ribonuclease-A--pyrimidine-nucleotide complexes. 299 93

The gene for Bacillus amyloliquefaciens extracellular RNase (barnase) has been cloned in an inactive form in Escherichia coli following insertional mutagenesis by transposon Tn917. The nucleotide (nt) sequence of the gene was determined and the deduced amino acid (aa) sequence found to correspond almost precisely to the previously determined sequence. An open reading frame (ORF) of 72 codons precedes the mature sequence. The probable translation start site is 46 or 47 codons before the N-terminal alanine of the mature protein, 11 (or 14) bp from a putative ribosome-binding site (RBS). Within this leader sequence is a hydrophobic 15 aa core preceded by three basic residues which is characteristic of a secretory signal sequence. A pro-barnase protein with four extra aa at the N-terminus has been detected extracellularly indicating that the signal peptidase-cutting site lies before the mature protein. An inverted repeat that may act as a transcription terminator was found at the 3' end of the gene. The gene is maintained in E. coli with a short inverted repeat from the termini of Tn917 inserted into the coding sequence. Northern blot analysis of RNA from B. amyloliquefaciens shows an approx. 780-nt transcript produced during exponential and stationary growth phases. The inactive cloned gene produces an approx. 480-nt transcript in E. coli and two transcripts of approx. 480 and 780 nt in Bacillus subtilis.
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PMID:Cloning, sequencing and transcription of an inactivated copy of Bacillus amyloliquefaciens extracellular ribonuclease (barnase). 300 90

Recent experiments showed that a single base pair (G3:U70) in the amino acid acceptor helix is a major determinant for the identity of Escherichia coli alanine transfer RNA. Experiments reported here show that bound alanine tRNA synthetase protects (from ribonuclease attack) seven consecutive phosphodiester linkages on the 3'-side of the acceptor-T psi C helix (phosphates 65-71) and a few additional sites that are in scattered locations. There is no evidence for interaction of the enzyme with the anticodon, a sequence which can be varied without effect on recognition by alanine tRNA synthetase.
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PMID:Evidence for interaction of an aminoacyl transfer RNA synthetase with a region important for the identity of its cognate transfer RNA. 305 91

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

A procedure of large-scale isolation of homogeneous ribonuclease Th1 from cultural filtrates of Trichoderma harzianum with a yield over 50% has been developed. Three ion-exchange chromatographies on CM- and DEAE-cellulose gave 7500 fold purification of the protein with a specific activity of ca. 4500 U/mg. The RNase Th1 is shown to be a basic protein (pI 9.5) with Mr 10,747; it contains 106 amino acid residues (2 Asp, 6 Asn, 9 Thr, 12 Ser, 2 Glu, 1 Gln, 4 Pro, 16 Gly, 14 Ala, 4 Cys, 7 Val, 5 Ile, 2 Leu, 7 Tyr, 6 Phe, 2 His, 4 Lys, 3 Arg). The total amino acid sequence of RNase Th1 was determined and, on comparison with other guanyl-specific fungal RNases, showed a significant degree of homology, thus indicating probability of a common origin. By means of the equilibrium dialysis, crystals of RNase Th1 were obtained with the space group P3(2)21, a = b = 55.7, c = 80.1 A. A preliminary X-ray study of RNase Th1 was undertaken.
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PMID:[Isolation, analysis of amino acid sequence and crystallization of the extracellular ribonuclease Th1 from Trichoderma harzianum-01]. 313 1

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

The primary structures of the blood vessel inducing protein human angiogenin and human pancreatic ribonuclease (RNase) are 35% identical. Angiogenin catalyzes the limited cleavage of ribosomal RNA (18 and 28 S), yielding a characteristic pattern of polynucleotide products, but shows no significant activity toward conventional pancreatic RNase substrates [Shapiro, R., Riordan, J. F., & Vallee, B. L. (1986) Biochemistry 25, 3527-3532]. Angiogenin/RNase hybrid enzymes--wherein particular regions of primary structure in RNase are replaced by the corresponding segments of angiogenin--serve to explore the structural features underlying angiogenin's characteristic activities. Herein we show that synthetic angiogenin peptides, Ang(1-21) and Ang(108-123), form noncovalent complexes with inactive fragments of bovine RNase A--RNase(21-124) (i.e., S-protein) and RNase(1-118), respectively--with regeneration of activity toward conventional RNase substrates. Maximal activities for the Ang(1-21)/S-protein complex (Kd = 1.0 microM) are 52%, 45%, and 15% toward cytidine cyclic 2',3'-phosphate, cytidylyl(3'----5')adenosine, and yeast RNA, respectively. In contrast, activities of the RNase(1-118)/Ang(108-123) hybrid (Kd = 25 microM) are 1-2 orders of magnitude lower toward cyclic nucleotides and dinucleoside phosphates. However, substitution of phenylalanine for Leu-115 in Ang(108-123) increases activity up to 100-fold. Both His-13 and His-114 in the angiogenin peptides are required for activity since their substitution by alanine yields inactive complexes. Importantly, the pattern of polynucleotide products formed during cleavage of ribosomal RNA by the Ang(1-21)/S-protein hybrid shows a striking resemblance to that formed by angiogenin, demonstrating that the hybrid retains features of both angiogenin and RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enzymatically active angiogenin/ribonuclease A hybrids formed by peptide interchange. 334 27


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