Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study. 131 2

From calculations of a model reaction scheme for base-catalyzed RNA hydrolysis, a pentacoodinate dianionic intermediate 2a (Storer, et al., J. Am. Chem. Soc., 1991, 113, 5216-5219) as well as two transition states, TS1 and TS2, to the intermediate have been located by ab initio calculations at the 3-21G* level. Although the intermediate, which has the well depth on the order of kBT, is unlikely to be kinetically significant, the overall rate-limiting transition state structure TS2 obtained at 3-21G* level is very close to the corresponding structure at the STO-3G level; it has an extended P-O(5') bond breaking character. These gas-phase calculation results are used to qualitatively interpret mutagenesis results of Barnase and RNase T1 where water molecules are absent from the active site.
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PMID:RNA hydrolysis via an oxyphosphorane intermediate. 138 73

In order to study the inhibitory effect of Zn2+ on ribonuclease T1 [RNase T1; Itaya & Inoue (1982). Biochem. J. 207, 357-362], the enzyme was cocrystallized with 2 mM Zn2+, pH 5.2, from a solution containing 55% (v/v) 2-methyl-2,4-pentanediol. The crystals are orthorhombic, P2(1)2(1)2(1), a = 48.71 (1), b = 46.51 (1), c = 41.14 (1) A, Z = 4, V = 93203 A3. The crystal structure was determined by molecular replacement and refined by restrained least-squares methods based on Fhkl for 8291 unique reflections with Fo greater than or equal to 1 sigma (Fo) in the resolution range 10 to 1.8 A and converged at a crystallographic R factor of 0.140. The Zn2+ is not bonded to the active site of RNase T1, probably because the His40 and His92 side chains are protonated. Zn2+ occupies the same site as Ca2+ in a series of crystal structures of free and nucleotide-complexed RNase T1. It is coordinated to Asp15 carboxylate and to six water molecules forming a dodecahedron of square antiprismatic form. The Zn2+...O distances are approximately 2.5 A, suggesting that Zn2+ is clathrated and not coordinated, which would require distances of 2.0 A.
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PMID:Structure of ribonuclease T1 complexed with zinc(II) at 1.8 A resolution: a Zn2+.6H2O.carboxylate clathrate. 151 6

The structure of the Gln25 variant of ribonuclease T1 (RNase T1) crystallized at pH 7 and at high ionic strength has been solved by molecular replacement using the coordinates of the Lys25-RNase T1/2'-guanylic acid (2'GMP) complex at pH 5 [Arni et al. (1988) J. Biol. Chem. 263, 15358-15368] and refined by energy minimization and stereochemically restrained least-squares minimization to a crystallographic R-factor of 14.4% at 1.84-A resolution. The asymmetric unit contains three molecules, and the final model consists of 2302 protein atoms, 3 sulfates (at the catalytic sites), and 179 solvent water molecules. The estimated root mean square (rms) error in the coordinates is 0.15 A, and the rms deviation from ideality is 0.018 A for bond lengths and 1.8 degrees for bond angles. Significant differences are observed between the three molecules in the asymmetric unit at the base recognition and catalytic sites.
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PMID:Three-dimensional structure of Gln25-ribonuclease T1 at 1.84-A resolution: structural variations at the base recognition and catalytic sites. 155 99

Ribonuclease T1 was purified from an Escherichia coli overproducing strain and co-crystallized with adenosine 2'-monophosphate (2'-AMP) by microdialysis against 50% (v/v) 2-methyl-2,4-pentanediol in 20 mM sodium acetate, 2 mM calcium acetate, pH 4.2. The crystals have orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 48.93(1), b = 46.57(4), c = 41.04(2) A; Z = 4 and V = 93520 A3. The crystal structure was determined on the basis of the isomorphous structure of uncomplexed RNase T1 (Martinez-Oyanedel et al. (1991) submitted for publication) and refined by least squares methods using stereochemical restraints. The refinement was based on Fhkl of 7,445 reflections with Fo greater than or equal to 1 sigma (Fo) in the resolution range of 10-1.8 A, and converged at a crystallographic R factor of 0.149. The phosphate group of 2'-AMP is tightly hydrogen-bonded to the side chains of the active site residues Tyr38, His40, Glu58, Arg77, and His92, comparable with vanadate binding in the respective complex (Kostrewa, D., Choe, H.-W., Heinemann, U., and Saenger, W. (1989) Biochemistry 28, 7592-7600) and different from the complex with guanosine 2'-monophosphate (Arni, R., Heinemann, U., Tokuoka, R., and Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368) where the phosphate does not interact with Arg77 and His92. The adenosine moiety is not located in the guanosine recognition site but stacked on Gly74 carbonyl and His92 imidazole, which serve as a subsite, as shown previously (Lenz, A., Cordes, F., Heinemann, U., and Saenger, W. (1991) J. Biol. Chem. 266, 7661-7667); in addition, there are hydrogen bonds adenine N6H . . . O Gly74 (minor component of three-center hydrogen bond) and adenosine O5' . . . O delta Asn36. These binding interactions readily explain why RNase T1 has some affinity for 2'-AMP. The molecular structure of RNase T1 is only marginally affected by 2'-AMP binding. Its "empty" guanosine-binding site features a flipped Asn43-Asn44 peptide bond and the side chains of Tyr45, Glu46 adopt conformations typical for RNase T1 not involved in guanosine binding. The side chains of amino acids Leu26, Ser35, Asp49, Val78 are disordered. The disorder of Val78 is of interest since this amino acid is located in a hydrophobic cavity, and the disorder appears to be correlated with an "empty" guanosine-binding site. The two Asp15 carboxylate oxygens and six water molecules coordinate a Ca2+ ion 8-fold in the form of a square antiprism.
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PMID:Crystal structure of ribonuclease T1 complexed with adenosine 2'-monophosphate at 1.8-A resolution. 165 20

The crystal structures of ribonuclease from Streptomyces aureofaciens (RNase Sa) and its complex with 3'-guanylic acid (guanosine 3'-monophosphate, 3'-GMP) have been determined by the method of isomorphous replacement. The atomic parameters have been refined by restrained least-squares minimization using data in the resolution range 10.0-1.8 A. All protein atoms and more than 230 water atoms in the two crystal structures have been refined to crystallographic R factors of 0.172 and 0.175 respectively. The estimated r.m.s. error in the atomic positions ranges from 0.2 A for well-defined atoms to about 0.5 A for more poorly defined atoms. There are two enzyme molecules in the asymmetric unit, built independently, and referred to as molecules A and B. The value of the average B factor for protein atoms in both structures is about 19 A2 and for water molecules about 35 A2. Electron density for the substrate analogue 3'-GMP was found only at the active site of molecule A. The density was very clear and the positions of all 3'-GMP atoms were refined with precision comparable to that of the protein.
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PMID:Determination and restrained least-squares refinement of the structures of ribonuclease Sa and its complex with 3'-guanylic acid at 1.8 A resolution. 165 32

Hydrophobic effects on binding of ribonuclease T1 to guanine bases of several ribonucleotides have been proved by mutating a hydrophobic residue at the recognition site and by measuring the effect on binding. Mutation of a hydrophobic surface residue to a more hydrophobic residue (Tyr45----Trp) enhances the binding to ribonucleotides, including mononucleotide inhibitor and product, and a synthetic substrate-analog trinucleotide as well as the binding to dinucleotide substrates and RNA. Enhancements on binding to non-substrate ribonucleotides by the mutation have been observed with free energy changes ranging from -2.2 to -3.9 kJ/mol. These changes are in good agreement with that of substrate binding, -2.3 kJ/mol, which is calculated from Michaelis constants obtained from kinetic studies. It is shown, by comparing the observed and calculated changes in binding free energy with differences in the observed transfer free energy changes of the amino acid side chains from organic solvents to water, that the enhancement observed on guanine binding comes from the difference in the hydrophobic effects of the side chains of tyrosine and tryptophan. Furthermore, a linear relationship between nucleolytic activities and hydrophobicity of the residues (Ala, Phe, Tyr, Trp) at position 45 is observed. The mutation could not change substantially the base specificity of RNase T1, which exhibits a prime requirement for guanine bases of substrates.
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PMID:Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease T1. 172 96

In the high resolution (1.7-1.9 A) crystal structures of ribonuclease T1 (RNase T1) in complex with guanosine, guanosine 2'-phosphate, guanylyl 2',5'-guanosine, and vanadate, there are 30 water sites in nearly identical (+/- 1 A) positions that are considered conserved. One water is tightly bound to Asp76(O delta), Thr93(O gamma), Cys6(O), and Asn9(N); another bridges two loops by hydrogen-bonding to Tyr68(O eta) and to Ser35(N), Asn36(N); a loop structure is stabilized by two waters coordinated to Gly31(O) and His27(N delta), and by water bound to cis-Pro39(O). Most notable is a hydrogen-bonded chain of 10 water molecules. Waters 1-5 of this chain are inaccessible to solvent, are anchored at Trp59(N), and stitch together the loop formed by segments 60-68; waters 5-8 coordinate to Ca2+, and waters 9 and 10 hydrogen-bond to N-terminal side chains of the alpha-helix. The water chain and two conserved water molecules are bound to amino acids adjacent to the active site residues His40, Glu58, Arg77, and His92; they are probably involved in maintaining their spatial orientation required for catalysis. Water sites must be considered in genetic engineering; the mutation Trp59Tyr, which probably influences the 10-water chain, doubles the catalytic activity of RNase T1.
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PMID:Structurally conserved water molecules in ribonuclease T1. 190 May 11

The complex formed between ribonuclease T1 (RNase T1) and guanosine-3',5'-bisphosphate (3',5'-pGp) crystallizes in the cubic space group I23 with alpha = 86.47 (4) A. X-ray data were collected on a four-circle diffractometer to 3.2 A resolution and the structure was determined by molecular-replacement methods [ULTIMA; Rabinovich & Shakked (1984). Acta Cryst. A40, 195-200] based on the RNase T1 coordinates taken from the complex with guanosine-2'-phosphate. Refinement converged at 16.6% for 1540 data with Fo greater than 1 sigma (Fo) with acceptable stereochemistry. The RNase T1 conformation is comparable to that in other complexes which crystallize preferentially in space group P2(1)2(1)2(1) except for side chains that interact intermolecularly. The guanine of 3',5'-pGp is bound to the recognition site in the same way as in other guanine-containing complexes except for its interaction with Glu46. The side-chain carboxylate of this amino acid does not form hydrogen bonds to N1H and N2H of guanine but is rotated so as to permit insertion of two water molecules which replace its acceptor functions. In contrast to other guanosine derivatives which are bound to RNase T1 in the syn form, 3',5'-pGp is anti. This conformation positions the two phosphate groups 'outside' the protein, with hydrogen-bonding contacts only to water molecules; the active site is filled by water. The RNase T1-3',5'-pGp complex probably has biological significance as it may represent the enzyme-product complex before dissociation.
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PMID:X-ray analysis of cubic crystals of the complex formed between ribonuclease T1 and guanosine-3',5'-bisphosphate. 193 Aug 33

The enzyme ribonuclease T1 (RNase T1) isolated from Aspergillus oryzae was cocrystallized with the specific inhibitor guanylyl-2',5'-guanosine (2',5'-GpG) and the structure refined by the stereochemically restrained least-squares refinement method to a crystallographic R-factor of 14.9% for X-ray data above 3 sigma in the resolution range 6 to 1.8 A. The refined model consists of 781 protein atoms, 43 inhibitor atoms in a major site and 29 inhibitor atoms in a minor site, 107 water oxygen atoms, and a metal site assigned as Ca. At the end of the refinement, the orientation of His, Asn and Gln side-chains was reinterpreted on the basis of two-dimensional nuclear magnetic resonance data. The crystal packing and enzyme conformation of the RNase T1/2',5'-GpG complex and of the near-isomorphous RNase T1/2'-GMP complex are comparable. The root-mean-square deviation is 0.73 A between equivalent protein atoms. Differences in the unit cell dimensions are mainly due to the bound inhibitor. The 5'-terminal guanine of 2',5'-GpG binds to RNase T1 in much the same way as in the 2'-GMP complex. In contrast, the hydrogen bonds between the catalytic center and the phosphate group are different and the 3'-terminal guanine forms no hydrogen bonds with the enzyme. This poor binding is reflected in a 2-fold disorder of 2',5'-GpG (except the 5'-terminal guanine), which originates from differences in the pucker of the 5'-terminal ribose. The pucker is C2'-exo for the major site (2/3 occupancy) and C1'-endo for the minor site (1/3 occupancy). The orientation of the major site is stabilized through stacking interactions between the 3'-terminal guanine and His92, an amino acid necessary for catalysis. This might explain the high inhibition rate observed for 2',5'-GpG, which exceeds that of all other inhibitors of type 2',5'-GpN. On the basis of distance criteria, one solvent peak in the electron density was identified as metal ion, probably Ca2+. The ion is co-ordinated by the two Asp15 carboxylate oxygen atoms and by six water molecules. The co-ordination polyhedron displays approximate 4m2 symmetry.
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PMID:Three-dimensional structure of ribonuclease T1 complexed with guanylyl-2',5'-guanosine at 1.8 A resolution. 254 Dec 56


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