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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 present a calculation of the relative changes in binding free energy between the complex of
ribonuclease T1
(RNase Tr) with its
inhibitor 2
'-guanosine monophosphate (2'GMP) and that of
RNase T1
-2'-adenosine monophosphate (2'AMP) by means of a thermodynamic perturbation method implemented with molecular dynamics. Using the available crystal structure of the
RNase T1
-2'GMP complex, the structure of the
RNase T1
-2'AMP complex was obtained as a final structure of the perturbation calculation. The calculated difference in the free energy of binding (delta delta Gbind) was 2.76 kcal/mol. This compares well with the experimental value of 3.07 kcal/mol. The encouraging agreement in delta delta Gbind suggests that the interactions of inhibitors with the enzyme are reasonably represented. Energy component analyses of the two complexes reveal that the active site of
RNase T1
electrostatically stabilizes the binding of 2'GMP more than that of 2'AMP by 44 kcal/mol, while the van der Waals' interactions are similar in the two complexes. The analyses suggest that the mutation from Glu46 to Gln may lead to a preference of
RNase T1
for adenine in contrast to the guanine preference of the wild-type enzyme. Although the molecular dynamics equilibration moves the atoms of the
RNase T1
-2'GMP system about 0.9 A from their X-ray positions and the mutation of the G to A in the active site increases the deviation from the X-ray structure, the mutation of the A back to G reduces the deviation. This and the agreement found for delta delta Gbind suggest that the molecular dynamics/free energy perturbation method will be useful for both energetic and structural analysis of protein-ligand interactions.
...
PMID:Calculation of the relative binding free energy of 2'GMP and 2'AMP to ribonuclease T1 using molecular dynamics/free energy perturbation approaches. 215 20
The complex formed between the enzyme
ribonuclease T1
(
EC 3.1.27.3
) and its specific
inhibitor 2
'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of
ribonuclease T1
is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with
ribonuclease T1
, the
inhibitor 2
'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of
ribonuclease T1
(His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.
...
PMID:Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution. 284 11
Two mutants of
ribonuclease T1
(RNaseT1), [59-tyrosine]
ribonuclease T1
(W59Y) and [45-tryptophan,59-tyrosine]
ribonuclease T1
(Y45W/W59Y) possess between 150% and 190% wild-type activity. They have been crystallised as complexes of the
inhibitor 2
'-guanylic acid and analysed by X-ray diffraction at resolutions of 0.23 nm and 0.24 nm, respectively. The space group for both is monoclinic, P2(1), with two molecules/asymmetric unit, W59Y: a = 4.934 nm, b = 4.820 nm, c = 4.025 nm, beta = 90.29 degrees. Y45W/W59Y: a = 4.915 nm, b = 4.815 nm, c = 4.015 nm, beta = 90.35 degrees. Compared to wild-type RNaseT1 in complex with 2'-guanylic acid (2'GMP) both mutant inhibitor complexes indicate that the replacement of Trp59 by Tyr leads to a 0.04-nm inward shift of the single alpha-helix and to significant differences in the active-site geometry, inhibitor conformation and inhibitor binding. Calorimetric studies of a range of mutants [24-tryptophan]
ribonuclease T1
(Y24W), [42-tryptophan]
ribonuclease T1
(Y42W), [45-tryptophan]
ribonuclease T1
(Y45W), [92-alanine]
ribonuclease T1
(H92A) and [92-threonine]
ribonuclease T1
(H92T) with and without the further mutation Trp59-->Tyr showed that mutant proteins for which Trp59 is replaced by Tyr exhibit slightly decreased thermal stability.
...
PMID:X-ray crystallographic and calorimetric studies of the effects of the mutation Trp59-->Tyr in ribonuclease T1. 812 11
The function of the conserved Phe 100 residue of
RNase T1
(
EC 3.1.27.3
) has been investigated by site-directed mutagenesis and X-ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kcat reduced 75-fold and a small increase in Km for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild-type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala
RNase T1
complexed with the specific
inhibitor 2
'-GMP are restricted to the active site. Repositioning of 2'-GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.
...
PMID:A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1. 884 43
The
ribonuclease T1
variant 9/5 with a guanine recognition segment, altered from the wild-type amino acid sequence 41-KYNNYE-46 to 41-EFRNWQ-46, has been cocrystallised with the specific
inhibitor 2
'-GMP. The crystal structure has been refined to a crystallographic R factor of 0.198 at 2.3 A resolution. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 A, 2'-GMP is fixed to the primary recognition site due to increased aromatic stacking interactions. The phosphate group of 2'-GMP is located about 4.2 A apart from its position in wild-type
ribonuclease T1
-2'-GMP complexes, allowing a Ca(2+), coordinating this phosphate group, to enter the binding pocket. The crystallographic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine affinity and turnover rate. The presence of Ca(2+) was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent.
...
PMID:Structural analysis of an RNase T1 variant with an altered guanine binding segment. 1060 Mar 81
The backbone dynamics of free
ribonuclease T1
and its complex with the competitive
inhibitor 2
'GMP have been studied by (15)N longitudinal and transverse relaxation experiments, combined with {(1)H, (15)H} NOE measurements. The intensity decay of individual amide cross peaks in a series of ((1)H, (15)N)-HSQC spectra with appropriate relaxation periods (Kay, L.E. et al. (1989) Biochemistry, 28, 8972-8979; Kay, L.E. et al. (1992) J. Magn. Reson., 97, 359-375) was fitted to a single exponential by using a simplex algorithm in order to obtain (15)N T(1) and T(2) relaxation times. These experimentally obtained values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559; 4559-4570). The microdyramical parameters accessible by this approach clearly indicate a correlation between the structural flexibility and the tertiary structure of
ribonuclease T1
, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The results obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.
...
PMID:Backbone dynamics of ribonuclease T1 and its complex with 2'GMP studied by two-dimensional heteronuclear NMR spectroscopy. 2291 Nov 59
