Determination of platinated purines in oligoribonucleotides by limited digestion with ribonucleases T1 and U2.

Platinum complexes which are known to react preferentially with guanine (G) and adenine (A) bases of oligonucleotides can be used as tools to analyze their tertiary structures and eventually to cross-link them. However, this requires efficient methods to allow the identification and quantification of the corresponding adducts which have so far been developed only for oligodeoxyribonucleotides. Maxam-Gilbert type digestions cannot be used for RNAs and HPLC techniques would require too large amounts of expensive material for separation and further characterization. We report a method to determine platination sites on oligoribonucleotides based on the cleavage activity of ribonucleases T1 and U2. To test the method, these enzymes were first used under conditions of limited digestion on 5-mer oligoribonucleotides platinated at a single defined purine. The phosphodiester bond on the 3' side of platinated G or A appeared fully resistant to cleavage by ribonuclease T1 or U2, respectively. An inhibitory effect was also observed due to neighboring platinated purines, which decreases with their distance (-2, -1, +1, +2) from the cleavage site and with the enzyme concentration. The method allowed the identification and quantification of the platination sites of a 17-mer oligoribonucleotide, based on the analysis of the mixture of monoplatinated adducts.

Structural analysis of an RNase T1 variant with an altered guanine binding segment.

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.

Preference for syn conformation: crystal structures of free acid and ammonium salt of adenosine 2'-monophosphate: an inhibitor of RNase T1.

This paper reports the crystal structures of free acid and ammonium salt of adenosine 2'-monophosphate (2'-AMP). 2'-AMP crystallizes in the hexagonal space group P6(5)22 with a = 9.530(3) A, c = 73.422(2) A, and Z= 12. 2'-AMP.NH4 crystallizes in the trigonal space group P3(1) with a = 9.003(2) A, c = 34.743(2) A and Z= 6. Both the structures were solved by direct methods and refined by full matrix least- squares method to final R factors of 0.080 and 0.038 for 2'-AMP and 2'-AMP.NH4 respectively. The adenine bases of both the structures are in syn conformation contrasting with the anti geometry in 3'-AMP, 5'-AMP and the enzyme bound state. Ribose moiety of 2'-AMP is in C2' -endo conformation. However, the ribose moieties of both the nucleotide molecules display C2'-endo-C3'-exo twist conformation in 2'- AMP.NH4 structure. Both structures demonstrate g+ conformation about C4' -C5' bond. 2'-AMP and one of the nucleotide molecules of 2'-AMP.NH4 are protonated at N1 and the ammonium ion is involved in a bifurcated hydrogen bond with O3' B and O3A atoms. A characteristic feature of both the structures is the intramolecular O5' -N3 hydrogen bond. Our crystallographic results on 2'-AMP corroborates the earlier conclusion that the enzyme-bound state is not the lowest energy state of this nucleotide. 2' -AMP displays base-ribose 04' stacking not seen in the 2'-AMP.NH4 structure. Theoretical and experimental studies on 2'-, 3'- and 5'-AMP structures have been discussed.

A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1.

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.

Modes of mononucleotide binding to ribonuclease T1.

The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.

Structure of ribonuclease T1 complexed with zinc(II) at 1.8 A resolution: a Zn2+.6H2O.carboxylate clathrate.

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.

Synthesis and kinetic study of transition state analogs for ribonuclease T1.

Based on the proposal that ribonucleases cleave the RNA phosphodiester bond with a mechanism involving pentacovalent phosphorous as transition state, complexes of guanosine and inosine with vanadate-(IV, V), molybdate-(VI), tungstate-(VI), chromate-(VI) and hexacyanochromate-(III) were synthesized and probed as inhibitors of recombinant ribonuclease T1, obtained from an E. coli. overproducing strain. The apparent dissociation constants of these inhibitors and RNase T1, as determined by Michaelis-Menten kinetics, vary between 0.5-0.9 microM and indicate very strong binding, 100- to 1000-fold stronger than the binding of guanosine (Kd = 545 microM) and inosine (Kd = 780 microM), and 50-100-fold stronger than the binding of the product 3' GMP (Kd = 55 microM). Therefore the synthesized inhibitors may be considered as genuine transition state analogs for the enzyme.

Raman spectroscopic study on the structure of ribonuclease F1 and the binding mode of inhibitor.

The structure of RNase F1 in aqueous solution has been studied by Raman spectroscopy and compared with that of a homologous enzyme, RNase T1. RNase F1 contains less beta-sheet and alpha-helical structure and more irregular structure than RNase T1. The strength of hydrogen bonding is weak in the beta-sheet and strong in the alpha-helix compared to that of RNase T1. Two disulfide bridges take the gauche-gauche and gauche-trans conformations, respectively. The overall hydrogen bonding of nine Tyr side chains in RNase F1 is very similar to that in RNase T1. Both of two His residues have pKa values around 8.2, which are close to those of the His residues in the active site of RNase T1. Upon binding of 2'-GMP, the hydrogen bonding of some Tyr side chains changes to a more proton-donating state. 2'-GMP is strongly hydrogen bonded with the enzyme at N7 of the guanine ring and takes the C3' endo-syn conformation. The binding mode of the inhibitor is identical to that found for RNase T1. In spite of significant differences in secondary structure, the molecular architecture of the active site seems to be highly conserved.

Thermodynamic analysis of the equilibrium, association and dissociation of 2'GMP and 3'GMP with ribonuclease T1 at pH 5.3.

Fluorescence titrations and temperature-jump relaxation experiments were performed as a function of temperature on ribonuclease T1 with the inhibitors 2'GMP and 3'GMP to obtain information on the energetics and molecular events controlling the binding of those inhibitors. Results from the titration and temperature-jump experiments were in agreement concerning the equilibrium constant. The larger equilibrium constant for 2'GMP is enthalpic in origin and is due to both a higher on rate and a lower off rate as compared to 3'GMP. On rates for both inhibitors appear to be below the diffusion controlled limit, apparently due to conformational changes in the portion of the active site responsible for recognition of the guanine base. Comparison of the measured enthalpic and entropic terms associated with the equilibrium constant determined from the fluorescence titrations are in disagreement with those calculated from the on and off rates indicating the presence of an induced conformational change in the 2'GMP-enzyme complex. This second conformational change appears to be due to additional interactions between 2'GMP and the catalytic portion of the active site, which may also be responsible for the differences in the binding constant, the on rate and the off rate between 2'GMP and 3'GMP.

Relative binding free energy calculations of inhibitors to two mutants (Glu46----Ala/Gln) of ribonuclease T1 using molecular dynamics/free energy perturbation approaches.

We present free energy perturbation calculations on the complexes of Glu46----Ala46 (E46A) and Glu46----Gln46 (E46Q) mutants of ribonuclease T1 (RNaseT1) with inhibitors 2'-guanosine monophosphate (GMP) and 2'-adenosine monophosphate (AMP) by a thermodynamic perturbation method implemented with molecular dynamics (MD). Using the available crystal structure of the RNaseT1-GMP complex, the structures of E46A-GMP and E46Q-GMP were model built and equilibrated with MD simulations. The structures of E46A-AMP and E46Q-AMP were obtained as a final structure of the GMP----AMP perturbation calculation respectively. The calculated difference in the free energy of binding (delta delta Gbind) was 0.31 kcal/mol for the E46A system and -1.04 kcal/mol for the E46Q system. The resultant free energies are much smaller than the experimental and calculated value of approximately 3 kcal/mol for the native RNaseT1, which suggests that both mutants have greater relative adenine affinities than native RNaseT1. Especially E46Q is calculated to have a larger affinity for adenine than guanine, as we suggested previously from the calculation on the native RNaseT1. Thus, the molecular dynamics/free energy perturbation method may be helpful in protein engineering, directed toward increasing or changing the substrate specificity of enzymes.

Binding modes of inhibitors of ribonuclease T1 as elucidated by analysis of two-dimensional NMR.

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP, or 5'GMP were assigned to specific amino acid residues by analyses of the two-dimensional NMR spectra in comparison with the crystal structure of the RNase T1-2'GMP complex. These assignments were subsequently correlated to those of free RNase T1 [Hoffmann & Rüterjans (1988) Eur. J. Biochem. 177, 539-560]. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced, which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take the syn form as a bound conformation, while 5'GMP takes the anti conformation around glycosidic bonds [Inagaki et al. (1985) Biochemistry 24, 1013-1020]. The slow-exchange process between free and bound forms involving Tyr42 and Tyr45 was found to be specific to the recognition of the guanine base.

Binding modes of inhibitors to ribonuclease T1 as elucidated by the analysis of two-dimensional NMR.

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP or 5'GMP were assigned to specific amino acid residues by 2D-NMR spectra in comparison with the crystal structure of RNase T1-2'GMP complex. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take syn form as a bound conformation, while 5'GMP takes anti conformation around glycosidic bonds.

Hexacyanochromate ion as a paramagnetic anion probe for active sites of enzymes.

Hexacyanochromate ion, (Cr(CN)6)3-, was applied to ribonuclease T1 (RNase T1), which specifically cleaves RNA chains at guanylic acid residues. From kinetic studies, this anion was shown to bind to the active site of RNase T1 as a competitive inhibitor. Therefore, the line broadening effect of NMR resonances due to binding of (Cr(CN)6)3- was analyzed for the mapping of the active site of RNase T1. His-40 C2 proton resonance was significantly broadened, following His-92 C2 proton resonance upon binding of (Cr(CN)6)3-, while His-27 C2 proton resonance did not show any appreciable line broadening. Moreover, from the pH dependence of the line broadening effect, the binding of (Cr(CN)6)3- was shown to be controlled by the ionic state of Glu-58. Based on the present NMR results and x-ray crystal structure, the active site structure of RNase T1 is discussed.

Binding modes of inhibitors to ribonuclease T1 as studied by nuclear magnetic resonance.

The binding modes of inhibitors to ribonuclease T1 (RNase T1) were studied by the analyses of 270-MHz proton NMR spectra. The chemical shift changes upon binding of phosphate, guanosine, 2'-GMP, 3'-GMP, 5'-GMP, and guanosine 3',5'-bis(phosphate) were observed as high field shifted methyl proton resonances of RNase T1. One methyl resonance was shifted upon binding of phosphate and guanosine nucleotides but not upon binding of guanosine. Four other methyl resonances were shifted upon binding of guanosine and guanosine nucleotides but not upon binding of phosphate. From the analyses of nuclear Overhauser effects for the pair of H8 and H1' protons, together with the vicinal coupling constants for the pair of H1' and H2' protons, the conformation of the guanosine moiety as bound to RNase T1 is found to be C3'-endo-syn for 2'-GMP and 3'-GMP and C3'-endo-anti for 5'-GMP and guanosine 3',5'-bis(phosphate). These observations suggest that RNase T1 probably has specific binding sites for the guanine base and 3'-phosphate group (P1 site) but not for the 5'-phosphate group (PO site) or the ribose ring. The weak binding of guanosine 3',5'-bis(phosphate) and 5'-GMP to RNase T1 is achieved by taking the anti form about the glycosyl bond. The productive binding to RNase T1 probably requires the syn form of the guanosine moiety of RNA substrates.

NMR studies on interactions of ribonuclease Sa with Guo-3'-P.

Some features of the interaction of guanyloribonuclease Sa from Streptomyces aureofaciens with its competitive inhibitor Guo-3'-P were investigated by 1H and 31P NMR spectroscopy. The pH dependence of chemical shifts of C(2)-H protons of the histidine residue of the enzyme were analysed, in the absence and presence of Guo-3'-P. This analysis showed that only one of the two histidines of ribonuclease Sa is located in the active site of the enzyme. 31P NMR resonances of the nucleotide and of its complex with the enzyme indicated that this histidine interacts with the phosphate group of the substrate. The possible relationship between the observed perturbation of the NMR titration curve of the active site of histidine and a conformational change in the enzyme molecule at a pH of approximately 7.5 is also discussed.

Steady-state kinetic studies of the inhibitory action of Zn2+ on ribonuclease T1 catalysis.

The kinetic mechanism of specific inhibition by Zn2+ of ribonuclease T1 catalysis was studied by steady-state kinetic analysis of transphosphorylation of dinucleotides, GpCp(3'), GpUp(2') and GpUp(3'), and dinucleoside monophosphates, GpC and GpU. The inhibition was not simply competitive, non-competitive or uncompetitive, but the kinetic data were compatible with a mechanism of 'fully mixed inhibition' in which a fully non-competitive action was associated with a partially competitive action. Apparent equilibrium quotients involved in this model of inhibition were determined for the dinucleotide substrates, and we found that binding of either of Zn2+ and substrate was facilitated when the other was bound. The location of Zn2+ was suggested to be near His-40 and/or His-92 of the ribonuclease T1 molecule.

[Antitumor action of RNAses modified by covalent binding with dextran m-aminobenzylhydroxymethyl ether]. / Protivoopukholevoe deistvie RNK-az, modifitsirovannykh kovalentnym sviazyvaniem s m-aminobenziloksimetilovym éfirom dekstrana.

Antitumor effect of pancreatic RNase and RNase from Actinomyces rimosus, as well as of their derivatives modified by dextran m-aminobenzylhydroxymethyl ether under different conditions was studied and compared. It was found that the efficacy of actinomycetous enzyme and its modified derivatives was superior to that of the analogous preparations of pancreatic RNase. Antitumor effect of the modified enzymes was higher than that of the native ones and depended on the modification conditions. It is concluded that biological efficacy of the RNases is determined by their origin and physico-chemical properties.

The structure and function of ribonuclease T1 XXIV. Preparation and properties of a stable water-insoluble polyacrylamide derivative of ribonuclease T1.

Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme.