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

---

Query: EC:3.1.27.3 (RNase T1)
1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The free form of ribonuclease T1 (RNase T1) has been crystallized at neutral pH, and the three-dimensional structure of the enzyme has been determined at 1.5 A nominal resolution. Restrained least-squares refinement yielded an R value of 14.3% for 12,623 structure amplitudes. The high resolution of the structure analysis permits a detailed description of the solvent structure around RNase T1, the reliable rotational setting of several side-chain amide and imidazole groups and the identification of seven disordered residues. Among these, the disordered and completely internal Val78 residue is noteworthy. In the RNase T1 crystal structures determined thus far it is always disordered in the absence of bound guanosine, but not in its presence. A systematic analysis of hydrogen bonding reveals the presence in RNase T1 of 40 three-center and an additional seven four-center hydrogen bonds. Three-center hydrogen bonds occur predominantly in the alpha-helix, where their minor components close 3(10)-type turns, and in beta-sheets, where their minor components connect the peptide nitrogen and carbonyl functions of the same residue. The structure of the free form is compared with complexes of RNase T1 with filled base recognition site and/or catalytic site. Several structural rearrangements occurring upon inhibitor or substrate binding are clearly apparent. In conjunction with the available biochemical knowledge, they are used to describe probable steps occurring early during RNase T1-catalyzed phosphate transesterification.
...
PMID:Ribonuclease T1 with free recognition and catalytic site: crystal structure analysis at 1.5 A resolution. 196 Jul 30

The three-dimensional structures of ribonuclease (RNase) T1 complexes with the inhibitors 2'-guanylic acid (2'-GMP), 3'-guanylic acid (3'-GMP), and 5'-guanylic acid (5'-GMP) were predicted by energy minimization studies. It is shown that these inhibitors can bind to RNase T1 in either of the ribose puckered conformations (C2'-endo and C3'-endo) in solid state and exist in significant amounts in both forms in solution. These studies are in agreement with the x-ray crystallographic studies of the 2'-GMP-Lys25-RNase T1 complex, where the inhibitor binds in C2'-endo puckered conformation. These results are also in good agreement with the available 1H-nmr results of Inagaki et al. [(1985) Biochemistry 24, 1013-1020], but differ from their conclusions where the authors favor only the C3'-endo ribose conformation for all the three inhibitors. The calculations explain the apparent discrepancies in the conclusions drawn by x-ray crystallographic and spectroscopic studies. An extensive hydrogen-bonding scheme was predicted in all the three complexes. The hydrogen-bonding scheme predicted for the 2'-GMP (C2'-endo)-RNase T1 complex agrees well with those reported from x-ray crystallographic studies. In all three complexes the base and the phosphate bind in nearly identical sites independent of the position of the phosphate or the ribose pucker. The glycosyl torsion angle favors a value in the +syn range in the 2'-GMP (C2'-endo)-RNase T1, 3'-GMP (C2'-endo)-RNase T1, and 3'-GMP (C3'-endo)-RNase T1 complexes; in the high-syn range in the 2'-GMP (C3'-endo)-RNase T1 complex; and in the -syn range in the 5'-GMP (C2'-endo)-RNase T1 and 5'-GMP (C3'-endo)-RNase T1 complexes. These results are in agreement with experimental studies showing that the inhibitory power decreases in the order 2'-GMP greater than 3'-GMP greater than 5'-GMP, and they also explain the high pKa value observed for Glu58 in the 2'-GMP-RNase T1 complex.
...
PMID:Computer modeling studies of ribonuclease T1-guanosine monophosphate complexes. 217 61

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.
...
PMID:Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles. 224 51

In the cocrystal formed by 7-methylguanosine-5'-phosphate.phenylalanine.6H2O, the interactions between guanine and phenylalanine are similar to those observed in the complex of ribonuclease T1 with 2'-guanylic acids, and those of the two G-proteins, Elongation Factor-Tu and ras oncogene p21, with GDP. They are similar in the following three points: (a) guanine N(1)H and N(2)H donate cyclic N-H...O hydrogen bonds to the carboxylate group of phenylalanine in the former cocrystal and to the side chain carboxylate group of Asp or Glu in the latter proteins, (b) O(6) of guanine accepts hydrogen bond(s) from main-chain NH group(s), and (c) the purine moiety is sandwiched between aromatic (or hydrophobic) amino acid side chains.
...
PMID:The specific guanine binding site of the ribonuclease T1 family enzymes and of G-proteins is modeled in the cocrystal formed by 7-methylguanosine-5'-phosphate and phenylalanine. 250 38

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.
...
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.
...
PMID:Refined X-ray structure of the low pH form of ribonuclease T1-2'-guanylic acid complex at 1.9 A resolution. 283 79

Molecular dynamics simulations in vacuum and with a water sphere around the active site were performed on the 2'GMP-RNase T1 complex. The presence of water led to the maintenance of the 2'-GMP-RNase T1 interactions as compared to the X-ray structure, including the hydrogen bonds implicated in the enzyme-inhibitor recognition process. The sidechain of His92 in the molecular dynamics water simulation, however, hydrogen bonds directly to the phosphate of 2'GMP in contrast to the X-ray structure but in support of the role of that residue in the enzyme's catalytic mechanism. Fluctuations of active-site residues are not strongly influenced by water, possibly owing to the exclusion of water by the bound 2'GMP, which did show an increase in mobility. Analysis of the 2'GMP-RNase T1 interactions versus time reveal an equilibrium fluctuation in the presence of water, leading to a less favorable 2'GMP-RNase T1 interaction energy, suggesting a possible relationship between picosecond fluctuations and inhibitor dissociation occurring in the millisecond time domain.
...
PMID:Molecular dynamics simulations of ribonuclease T1. Effect of solvent on the interaction with 2'GMP. 285 69

Wheat germ has been chosen as a representative eukaryote for study of ribosomal 5S RNA secondary structure. Proton homonuclear Overhauser enhancements (NOE's) at 500 MHz for the hydrogen-bonded base-pair protons in the 10-15 ppm region are used to establish the identity (A X U, G X C, or G X U) and base-pair sequence (e.g., G X C-A X U-C X G) within a given helical segment. Assignment of that segment to particular base pairs in the secondary structure is based upon NOE's conducted at different temperatures (to determine which signals "melt" together), variation of salt conditions (to produce differential chemical shifts in order to better distinguish components of an unresolved spectral envelope), and isolation and purification of RNase T1 cleavage fragments (in order to reduce the spectrum to just a few base pairs). The NOE patterns for the RNase T1 fragments are the same as in the intact 5S RNA, supporting the assumption that structural features of this region in the intact 5S RNA are preserved in the fragment. Chemical shifts predicted from ring current induced effects for a proposed base-pair sequence are then compared to experimental chemical shifts. By these methods, a portion of the "tuned helix" segment (namely, the base-pair sequence C18G60-A19U59-C20G58) is demonstrated spectroscopically for the first time in any 5S RNA. The tuned helix and common arm segments are less stable than the rest of the molecule. Variation of sodium and magnesium levels reveals multiple configurations of the wheat germ 5S RNA in solution.
...
PMID:Identification and assignment of base pairs in the "tuned helix" of intact and ribonuclease T1 cleavage fragments of wheat germ ribosomal 5S RNA via 500-MHz proton homonuclear Overhauser enhancements. 308 17

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.
...
PMID:Glu 46 of ribonuclease T1 is an essential residue for the recognition of guanine base. 312 58

Molecular dynamics simulations were performed on ribonuclease T1 (RNase T1; EC 3.1.27.3) to determine a structure for the free enzyme. Simulations starting with the X-ray coordinates for the 2'GMP-RNase T1 complex were done in vacuo and with an 18-A water ball around the active site using stochastic boundary conditions to understand the influence of water on both the structure and fluctuations of the enzyme. Removal of 2'GMP caused structural changes in the loop regions, including those directly interacting with the bound inhibitor in the crystal structure, while regions of secondary structure were less affected. The presence of solvent in the simulation damped the structural changes observed, which may be related to the use of full charges in both simulations. Fluctuations were also affected by the water, which generally increased both at the surface and in the interior of the protein. The active site in vacuo collapsed upon itself, forming a number of protein-protein hydrogen bonds leading to larger structural changes and lowered fluctuations while the presence of water kept the active site open, minimized structural changes, and increased fluctuations. Such fluctuations in the active site may be important for the binding of inhibitors or substrates to the enzyme. Lastly, results from the water simulation allow the prediction of a motion for a hypothetical tryptophan at position 45, which can ultimately be tested experimentally via time-resolved fluorescence using a site-specific mutant of the enzyme.
...
PMID:Molecular dynamics simulations of ribonuclease T1: analysis of the effect of solvent on the structure, fluctuations, and active site of the free enzyme. 313 27


<< Previous 1 2 3 4 5 6 7 8 9 Next >>

---