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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Angiogenin is a protein belonging to the superfamily of RNase A. The RNase activity of this protein is essential for its angiogenic activity. Although members of the RNase A family carry out RNase activity, they differ markedly in their strength and specificity. In this paper, we address the problem of higher specificity of angiogenin towards cytosine against uracil in the first base binding position. We have carried out extensive nano-second level molecular dynamics(MD) computer simulations on the native bovine angiogenin and on the CMP and UMP complexes of this protein in aqueous medium with explicit molecular solvent. The structures thus generated were subjected to a rigorous free energy component analysis to arrive at a plausible molecular thermodynamic explanation for the substrate specificity of angiogenin.
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PMID:A molecular dynamics study based post facto free energy analysis of the binding of bovine angiogenin with UMP and CMP ligands. 1156 27

Ribonucleases (RNases) possess a variety of biological activities and, under certain conditions, are deleterious. Hence, design of selective inhibitors has been suggested as a strategy for treating RNase-related disorders. In the present study, isothermal titration calorimetry was used to measure ion effects on binding thermodynamics of the RNase A competitive inhibitor 2'-CMP as a representative system. The reaction cell (37 degrees C) contained dialyzed RNase A (0.04-0.05 mM) in buffered solution (pH 5.5) of 50 mM Na(+), K(+), Ca(2+), or Mg(2+) acetate, verified spectrophotometrically. Thirty-five sequential injections (4 microl each, 3 min apart) were made of 2'-CMP (1.2 mM) in ion-matching buffer. The data were corrected for heat of dilution. There was a 1:1 interaction in each case. The estimated parameters (+/-S.D.) were: K(d) = 4.84 +/- 0.29 microM (Na(+)); 5.62 +/- 0.98 microM (K(+)); 24.44 +/- 6.96 microM (Ca(2+)); 28.74 +/- 0.43 microM (Mg(2+)); DeltaG(o) = -7.541 +/- 0.037 kcal/mol (Na(+)); -7.458 +/- 1.03 kcal/mol (K(+)); -6.574 +/- 0.173 kcal/mol (Ca(2+)); -6.442 +/- 0.009 kcal/mol (Mg(2+)); DeltaH(o) = -22.357 +/- 1.189 kcal/mol (Na(+)); -21.917 +/- 0.891 kcal/mol (K(+)); -20.223 +/- 1.503 kcal/mol (Ca(2+)); -26.570 +/- 1.579 kcal/mol (Mg(2+)); and DeltaS(o) = -0.048 +/- 0.004 kcal/mol-K (Na(+)); -0.047 +/- 0.003 kcal/mol-K (K(+)); -0.044 +/- 0.005 kcal/mol-K (Ca(2+)); -0.065 +/- 0.005 kcal/mol-K (Mg(2+)). Thus, all reactions were enthalpy-driven. Despite a 5-fold difference in K(d) between mono- and divalent ions, the ratio of ion hydration DeltaG(o) to K(d) was constant. These data should be useful for molecular modeling and suggest that inhibitor activity will be a function of cellular conditions (normal or pathological).
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PMID:Toward the design of ribonuclease (RNase) inhibitors: ion effects on the thermodynamics of binding of 2'-CMP to RNase A. 1202 20

Certain ribonucleases (RNases), such as eosinophil-derived neurotoxin, are associated with pathological conditions (e.g. asthma and inflammatory bowel disease) and can even be overtly cyto(neuro)toxic. It has been proposed that small-molecule inhibitors should have therapeutic utility. We used isothermal titration microcalorimetry to characterize reversible inhibitor cytidine 2'-monophosphate (2'-CMP) binding to RNase-A in a multi-ion buffer at 37 degrees as a representative system. The estimated parameters were: K(d)=13.9 microM; DeltaG degrees =-6.90 kcal/mol; DeltaH degrees =-15.7 kcal/mol; and DeltaS degrees =-0.028 kcal/mol-K ('enthalpy-driven' interaction). These data should assist drug design of small-molecule inhibitors of homologous RNase catalytic domains.
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PMID:Toward RNase inhibitors: thermodynamics of 2'-CMP/RNase-A binding in multi-ion buffer. 1209 69

The interaction of the dinucleotide inhibitor 5'-phosphothymidine(3',5')pyrophosphate adenosine 3'-phosphate (pTppAp) with bovine pancreatic ribonuclease A (RNase A) was characterized by calorimetry and solution NMR spectroscopy. Calorimetric data show that binding of pTppAp to RNase A is exothermic (DeltaH = -60.1 +/- 4.1 kJ/mol) with a dissociation constant of 16 nM at 298 K. At this temperature, the binding results in an entropy loss (TDeltaS = -16.8 +/- 7.3 kJ/mol) that is more favorable than that with the product analogue, 2'-CMP (TDeltaS = -31.3 +/- 0.9 kJ/mol). Temperature-dependent calorimetric experiments give a DeltaC(p) for ligand binding of -230 +/- 100 J/mol K. Binding of pTppAp results in noticeable effects on the backbone amide chemical shifts and dynamics. Amide backbone (15)N NMR spin-relaxation studies were performed on both apo RNase A and RNase A/pTppAp as a function of temperature. At each temperature, the model-free-determined order parameters, S(2), were significantly higher for RNase A/pTppAp than for the apo enzyme indicating a decrease in the conformational entropy of the protein upon ligand binding. Furthermore, the magnitude of this difference varies along the amino acid sequence specifically locating the entropic changes. The temperature dependence of S(2) at each residue enabled assessment of the local heat capacity changes (DeltaC(p)) from ligand binding. In an overall, average sense, DeltaC(p) for the protein backbone, determined from the NMR dynamics measurements, did not differ between apo RNase A and RNase A/pTppAp indicating that backbone dynamics contribute little to DeltaC(p) for protein-ligand interactions in this system. However, residue-by-residue comparison of the temperature-dependent change in entropy (DeltaS(B)) between free and bound forms reveals nonzero contributions to DeltaC(p) at individual sites. The balance of positive and negative changes reveals a redistribution of energetics upon binding. Furthermore, experiment and semiempirical estimates suggest that a large negative DeltaC(p) should accompany binding of pTppAp, and we conclude that this contribution must arise from factors other than amide backbone dynamics.
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PMID:Temperature dependence of the backbone dynamics of ribonuclease A in the ground state and bound to the inhibitor 5'-phosphothymidine (3'-5')pyrophosphate adenosine 3'-phosphate. 1273 69

Electrospray ionization mass spectrometry (ESI-MS) has proven to be an extremely powerful tool for studying biomolecular structures and noncovalent interactions. Here we report a method using a fully automated, chip-based nanoESI-MS system to determine the dissociation constants (Kd) for the complexes of two different proteins with their ligands. The automated nanoelectrospray system, consisting of the NanoMate and ESI chip, serves functionally as a combination of autosampler and nanoelectrospray ionization source. This system provides all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without carryover. The automated nanoESI system was used to investigate quantitative noncovalent interactions between ribonuclease A (RNase A) and cytidylic acid ligands (2'-CMP, CTP), a well-characterized model protein-ligand complex, and between an inactive endocellulase mutant (Thermobifida fusca Cel6A D117Acd) and four oligosaccharide ligands (cellotriose, cellotetraose, cellopentaose, cellohexaose). Both titration and competitive binding approaches were performed prior to automated nanoESI-MS analysis with a Q-TOF mass spectrometer. Dissociation constants for each complex were calculated from the sum of ion peak areas of free and complexed proteins during the titration and competition experiments. The measured Kd values for the RNase A-CMP and Cel6A D117Acd-G3 complexes were found to be in excellent agreement with the available published values obtained by standard spectroscopic titration techniques. To our knowledge, this is the first report of using an ESI-MS approach to study the interactions between a cellulase and oligosaccharides. The results provide new insights for understanding the nature of cellulase-cellulose interactions.
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PMID:Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry. 1296 45

Pharmacologic ligand-macromolecule interactions are commonly characterized by affinity (dissociation) constants such as K(d) or K(i) without regard to the protonation effect of the buffer used in the measurement. The protonation effect is demonstrated here using isothermal titration microcalorimetry measurements of the competitive inhibitor binding of cytidine 2'-monophosphate (2'-CMP) to RNase-A as a model system in buffers of different ionization Delta H(buffer). The results demonstrate the importance of protonation in measures of affinity.
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PMID:Protonation effect on drug affinity. 1472 24

A new ultrasensitive differential scanning calorimeter (DSC) instrument is described, which utilizes autosampling for continuous operation. High scanning rates to 250 deg/h with rapid cooling and equilibration between scans facilitates higher sample throughput up to 50 samples during each 24 h of unattended operation. The instrument is suited for those pharmaceutical applications where higher throughput is important, such as screening drug candidates for binding constant or screening solution conditions for stability of liquid protein formulations. Results are presented on the binding of five different anionic inhibitors to ribonuclease A, which included cytidine 2'-monophosphate (2'CMP), 3'CMP, uridine 3'-monophosphate, pyrophosphate, and phosphate. Binding constants K(B) (or dissociation constants K(d)) are obtained from the shift in the transition temperature T(M) for ribonuclease thermal unfolding in the presence of ligand relative to the transition temperature in the absence of ligand. Measured binding constants ranged from 155 M(-1) (K(d) = 6.45 mM) for the weak-binding phosphate anion to 13100 M(-1) (K(d) = 76.3 microM) for the strongest binding ligand, 2'CMP. The DSC method for measuring binding constants can also be extended to ultratight interactions involving either ligand-protein or protein-protein binding.
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PMID:An autosampling differential scanning calorimeter instrument for studying molecular interactions. 1509 Jan 59

In the age of biochemical systems biology, proteomics, and high throughput methods, the thermodynamic quantification of cytoplasmatic reaction networks comes into reach of the current generation of scientists. What is needed to efficiently extract the relevant information from the raw data is a robust tool for evaluating the number and stoichiometry of all observed reactions while providing a good estimate of the thermodynamic parameters that determine the molecular behavior. The recently developed phase-diagram method, strictly speaking a graphical representation of linkage or Maxwell Relations, offers such capabilities. Here, we extend the phase diagram method to nonideal conditions. For the sake of simplicity, we choose as an example a reaction system involving the protein RNase A, its inhibitor CMP, the osmolyte urea, and water. We investigate this system as a function of the concentrations of inhibitor and osmolyte at different temperatures ranging from 280 K to 340 K. The most interesting finding is that the protein-inhibitor binding equilibrium depends strongly on the urea concentration--by orders-of-magnitude more than expected from urea-protein interaction alone. Moreover, the m-value of ligand binding is strongly concentration-dependent, which is highly unusual. It is concluded that the interaction between small molecules like urea and CMP can significantly contribute to cytoplasmic nonideality. Such a finding is highly significant because of its impact on renal tissue where high concentrations of cosolutes occur regularly.
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PMID:Protein phase diagrams II: nonideal behavior of biochemical reactions in the presence of osmolytes. 1702 44

The conditions for accurately determining distance constraints from TrNOESY data on a small ligand (3'CMP) bound to a small protein (RNase A, <14 kDa) are described. For small proteins, normal TrNOESY conditions of 10:1 ligand:protein or greater can lead to inaccurate structures for the ligand-bound conformation due to the contribution of the free ligand to the TrNOESY signals. By using two ligand:protein ratios (2:1 and 5:1), which give the same distance constraints, a conformation of 3'CMP bound to RNase A was determined (glycosidic torsion angle, chi=-166 degrees ; pseudorotational phase angle, 0 degrees < or = P < or =36 degrees ). Ligand-protein NOESY cross peaks were also observed and used to dock 3'CMP into the binding pocket of the apo-protein (7rsa). After energy minimization, the conformation of the 3'CMP:RNase A complex was similar to the X-ray structure (1 rpf) except that a C3'-endo conformation for the ribose ring (rather than C2'-exo conformation) was found in the TrNOESY structure.
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PMID:Conformation of 3'CMP bound to RNase A using TrNOESY. 1741 40

Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural homology of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl groups, suggested that phosphate ester recognition sites on proteins might exhibit significant affinity for nonenzymatically formed borate esters. (11)B NMR studies and activity measurements on ribonuclease A (RNase A) in the presence of borate and several cytidine analogs demonstrate the formation of a stable ternary RNase A.3'-deoxycytidine-2'-borate ternary complex that mimics the complex formed between RNase A and a 2'-cytidine monophosphate (2'-CMP) inhibitor. Alternatively, no slowly exchanging borate resonance is observed for a ternary RNase A, borate, 2'-deoxycytidine mixture, demonstrating the critical importance of the 2'-hydroxyl group for complex formation. Titration of the ternary complex with 2'-CMP shows that it can displace the bound borate ester with a binding constant that is close to the reported inhibition constant of RNase A by 2'-CMP. RNase A binding of a cyclic cytidine-2',3'-borate ester, which is a structural homolog of the cytidine-2',3'-cyclic phosphate substrate, could also be demonstrated. The apparent dissociation constant for the cytidine-2',3'-borate.RNase A complex is 0.8 mM, which compares with a Michaelis constant of 11 mM for cytidine-2',3'-cyclic phosphate at pH 7, indicating considerably stronger binding. However, the value is 1,000-fold larger than the reported dissociation constant of the RNase A complex with uridine-vanadate. These results are consistent with recent reports suggesting that in situ formation of borate esters that mimic the corresponding phosphate esters supports enzyme catalysis.
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PMID:Ternary borate-nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry. 1795 92


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