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)

We used harmonic-content frequency-domain fluorometry to determine the anisotropy decays of a variety of single tryptophan peptides and proteins. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. For each protein, and for each quencher, data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. The decrease in decay times produced by quenching allows measurements to an upper frequency limit of 2 GHz. The chosen proteins provided a range of exposures of the tryptophan residues to the aqueous phase, these being ACTH, monellin, Staphylococcus nuclease and ribonuclease T1, in order of decreasing exposure. Examination of indole and several small peptides demonstrates the resolution limitations of the measurements; a correlation time of 12 ps was measured for indole in methanol at 40 degrees C. Comparison of the anisotropy decays of gly-trp-gly with leu-trp-leu revealed stearic effects of the larger leucine side chains on the indole ring. The anisotropy decay of gly-trp-gly revealed a 40 ps component for the indole side chain, which was resolved from the overall 150 ps correlation time of the tripeptide. Only the longer correlation time was observed for leu-trp-leu. With the exception of ribonuclease T1, each of the proteins displayed a subnanosecond component in the anisotropy decay which we assign to independent motions of the tryptophan residues. For example, Staphylococcus nuclease and monellin displayed segmental tryptophan motions with correlation times of 80 and 275 ps, respectively. The amplitudes of the rapid components increased with increasing exposure to the aqueous phase. These highly resolved anisotropy decays for proteins of known structure are suitable for comparison with molecular dynamic simulations.
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PMID:Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching. 164 47

In the crystal structure of the ribonuclease T1 (RNase T1; EC 3.1.27.3)-2'-GMP complex the hydrogen-bonding potential of the guanine base is saturated [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368]. The oxygens of the Glu46 carboxylate and the Asn98 main-chain carbonyl act as hydrogen-bond acceptors for the N(1)H-C(2)-N(2)H2 part of the base. We measured the transesterification kinetics of wild-type and Glu46Ala RNase T1 using the GpU, IpU, and XpU series of analogous substrates. We found that the N(1)H---Glu46 O epsilon 1, the N(2)H---Glu46 O epsilon 2, and the N(2)H---Asn98 O hydrogen bonds have an apparent contribution of 2.7, 1.1, and 1.2 kcal/mol to the interaction energy of the enzyme and the transition state of the substrate. Wild-type RNase T1 discriminates guanine from nonionized xanthine (a guanine analogue in which the exocyclic amino group is replaced by an oxygen) by about 4.4 kcal/mol. Loss of the specific hydrogen bonds with the exocyclic amino group of the guanine base accounts for 2.4 kcal/mol of this discrimination energy; 2.0 kcal/mol is due to unfavorable non-H-bonded oxygen-oxygen contacts in the enzyme-xanthine complex. A pH dependence study shows that the deprotonated form of xanthine (i.e., the 6-keto-2-enolate anion; pKa = 5.4) is far less preferred, if not excluded, as substrate by wild-type RNase T1; this may be attributed to an electrostatic repulsion of the negatively charged xanthine by the Glu46 carboxylate group.
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PMID:Quantitative analysis of the contribution of Glu46 and Asn98 to the guanosine specificity of ribonuclease T1. 189 29

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

The three-dimensional X-ray structure of the RNase T1[EC 3.1.27.3]-2'GMP complex crystallized at low pH value (4.0) was determined, and refined to 1.9 A resolution to give a final R value of 0.203. The refined model includes 781 protein atoms, 24 inhibitor atoms, and 43 solvent molecules. The imidazole rings of His27 and His40 interact with the carboxyl side chains of Glu82 and Glu58, respectively, whereas that of His92 is in contact with the main chain carbonyl oxygen of Ala75. In the complex, the ribose ring of the 2'GMP molecule adopts a C2'-endo puckering, and the exocyclic conformation is gauche(-)-gauche(+). The glycosyl torsion angle is in the syn range with an intramolecular hydrogen bond between N3 and O5', and the 2'-phosphate orientation is trans-gauche(-). The guanine base of the inhibitor is tightly bound to the base recognition site with five hydrogen bonds (N1--Glu46O epsilon 2, N2---Asn98O,O6---Asn44N, and N7 ---Asn43N delta 2/Asn43N) and is sandwiched between the phenolic ring portions of Tyr42 and Tyr45 by stacking interactions. The 2'-phosphate group interacts with Arg77N eta 2, Glu58O episilon 2, and Tyr 38O eta but not with any of the histidine residues. Arg77N eta 2 also interacts with Tyr38O eta. There is no interaction between the ribose moiety of the inhibitor and the enzyme.
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PMID:Refined X-ray structure of the low pH form of ribonuclease T1-2'-guanylic acid complex at 1.9 A resolution. 283 79

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.
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PMID:Rotational freedom of tryptophan residues in proteins and peptides. 684 81

An approach is described for extending free energy calculations to take into account the pH dependence of the relative binding of ligands to an enzyme or other receptor protein. The method is based on the calculation of the free energy difference for a single protonation state via the thermodynamic cycle simulation approach followed by inclusion of all possible protonation states of the enzyme and the inhibitor by use of a macroscopic continuum dielectric (Poisson-Boltzmann) model. A detailed formulation of the combined model is presented. It involves solution of the multiple equilibrium problem and makes use of the calculated pKa values of all titrating groups on both enzyme and ligand. The method is illustrated by calculations of the pH dependence of the differential binding of the inhibitors 2'GMP and 3'GMP to ribonuclease T1. A free energy simulation of the differential binding is made for a given protonation state of the enzyme and inhibitor. Although only qualitative agreement with experiment is obtained, the results provide insights concerning the interactions involved. The pH dependence of the binding is calculated by using the protonation state of the residues from the free energy simulation as the standard state for a Poisson-Boltzmann calculation. Information is obtained concerning the pKa values of the titrating amino acids in the free, 2'GMP and 3'GMP bound enzyme forms of RNase T1 and the difference in the pH dependence of the binding of 2'GMP and 3'GMP to RNase T1. The contributions of different types of interactions (e.g. protein residues versus solvent) to the free energy differences are examined. A free energy simulation of the pKa shift of Glu58 shows that it is important to consider both carboxyl oxygen atoms as possible protonation sites since they may behave very differently in a protein. It is found in the protein that the interactions with the solvent favor the neutral (protonated) state of Glu58. This contrasts sharply with the solution behavior, where the solvent favors the charged state. Analysis of the results shows that the interactions of bound water with other protein residues leads to the observed effect. Comparisons are made with a continuum calculation that uses the charged state employed in the free energy simulation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:pH dependence of binding reactions from free energy simulations and macroscopic continuum electrostatic calculations: application to 2'GMP/3'GMP binding to ribonuclease T1 and implications for catalysis. 772 31

The interactions of RNase A with cytidine 3'-monophosphate (3'-CMP) and deoxycytidyl-3',5'-deoxyadenosine (d(CpA)) were analyzed by X-ray crystallography. The 3'-CMP complex and the native structure were determined from trigonal crystals, and the d(CpA) complex from monoclinic crystals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein-inhibitor contacts are interpreted in terms of the catalytic mechanism. The general base His 12 interacts with the 2' oxygen, as does the electrostatic catalyst Lys 41. The general acid His 119 has 2 conformations (A and B) in the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3'-CMP complex. From the present structures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex permits a detailed analysis of the downstream binding site, which includes His 119 and Asn 71. The comparison of the present RNase A structures with an inhibitor complex of RNase T1 shows that there are important similarities in the active sites of these 2 enzymes, despite the absence of any sequence homology. The water molecules were analyzed in order to identify conserved water sites. Seventeen water sites were found to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the binding of the N-terminal helix to the rest of the protein and in the stabilization of the active site.
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PMID:The structures of RNase A complexed with 3'-CMP and d(CpA): active site conformation and conserved water molecules. 775 88

Glu58 is known to participate in phosphodiester transesterification catalyzed by the enzyme RNase T1. For Glu58 RNase T1, an altered mechanism has been proposed in which His40 replaces Glu58 as the base catalyst [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072]. Glu58Ala Rnase T1 has been cocrystallized with guanosine 2'-monophosphate (2'-GMP). The crystals are of space group P2(1), with one molecule per asymmetric unit (a = 32.44 A, b = 49.64 A, c = 26.09 A, beta = 99.17 degrees). The three-dimensional structure of the enzyme was determined to a nominal resolution of 1.9 A, yielding a crystallographic R factor of 0.178 for all X-ray data. Comparison of this structure with wild-type structures leads to the following conclusions. The minor changes apparent in the tertiary structure can be explained by either the mutation of Glu58 or by the change in the space group. In the active site, the extra space available through the mutation of Glu58 is occupied by the phosphate group (after a reorientation) and by a solvent molecule replacing a carboxylate oxygen of Glu58. This solvent molecule is a candidate for participation in the altered mechanism of this mutant enzyme. Following up on a study of conserved water sites in RNase T1 crystallized in space group P2(1)2(1)2(1) [Malin, R., Zielenkiewicz, P., & Saenger, W. (1991) J. Mol. Biol. 266, 4848-4852], we investigated the hydration structure for four different packing modes of RNase T1.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Crystallographic study of Glu58Ala RNase T1 x 2'-guanosine monophosphate at 1.9-A resolution. 790 40

Reduced oxygen tension (hypoxia) induces a 3-fold increase in stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. To investigate the possibility that RNA-protein interactions are involved in mediating this increase in stability, RNA gel shift assays were performed using different fragments of labeled TH mRNA and the S-100 fraction of PC12 cytoplasmic protein extracts. We identified a sequence within the 3'-untranslated region of TH mRNA that binds cytoplasmic protein. RNase T1 mapping revealed that the protein was bound to a 28 nucleotide long sequence that is located between bases 1551-1579 of TH mRNA. Moreover, protein binding to this fragment was prevented with an antisense oligonucleotide directed against bases 1551-1579 and subsequent RNase H digestion. This fragment of the 3'-untranslated region of TH mRNA is rich in pyrimidine nucleotides, and the binding of cytoplasmic protein to this fragment was reduced by competition with other polypyrimidine sequences including poly(C) but not poly(U) polymers. The binding of the protein to TH mRNA was increased when cytoplasmic proteins were extracted from PC12 cells exposed to hypoxia (5% O2) for 24 h. Electrophoresis of the UV cross-linked RNA-protein complex on SDS-polyacrylamide gel electrophoresis revealed a complex of 74 kDa. The potential role of this protein-TH mRNA interaction in regulation of TH mRNA stability during hypoxia is discussed.
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PMID:Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA. 790 89

The crystal structure of RNase T1 complexed with 3'-GMP has been determined. The glycosyl conformation of 3'-GMP is in the syn conformation, and the ribose adopts the O4'-endo pucker. This observed pucker is different from that in any complex structures of RNase T1. In the present complex, this energetically unfavorable conformation is stabilized by the water molecule with the bridged hydrogen bonds between the O2' and the O3' atoms of the ribose. The guanine base is recognized in the same manner as observed in the complex of 2'-GMP. The 2'-hydroxyl group of the ribose shows a tight hydrogen bond to both His-40 and Glu-58 with the suitable geometry for the proton transfer. These hydrogen bonds suggest that the two residues can participate directly in the proton transfer. His-92 is hydrogen bonded to two the proton transfer. His-92 is hydrogen bonded to two oxygen atoms of the phosphate group. Based on the geometry in the active site, the O1P atom may correspond to the O5' atom of the leaving nucleotide in the phosphoryl transfer or a water molecule as a nucleophile in the hydrolysis reaction. In the present complex, the conformations of the 3'-GMP molecule and the side chains of the catalytic residues would be represented as the conformation before the phosphoryl transfer reaction and/or after the hydrolysis reaction.
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PMID:Crystal structure of RNase T1 complexed with the product nucleotide 3'-GMP. Structural evidence for direct interaction of histidine 40 and glutamic acid 58 with the 2'-hydroxyl group of the ribose. 791 96


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