guanosine-monophosphate and 3--guanylic-acid

In [PtCl2(cis-1,4-DACH)] (DACH = diaminocyclohexane), the N-Pt-N bite angle (> or =97 degrees , as determined by X-ray diffraction analysis) is much larger than those found in other Pt complexes with bidentate diamines or in cisplatin (approximately 91 degrees ). Hence, the possibility exists that in (cis-1,4-DACH)PtG 2 adducts, rotation of the G's around the Pt-N7 bonds is slowed enough to allow observation of different conformers. In accord with this prevision, decreasing the temperature to 238 K enabled us to observe different conformers of (cis-1,4-DACH)Pt(5'-GMP) 2 (GMP = guanosine monophosphate). This observation is the first case in which such conformers for a platinum derivative with primary diamines and untethered guanines have been resolved and represents the closest model to clinically effective cisplatin obtained to date. We also found that the presence of the 1,4-DACH ligand increased the intensity of the circular dichroism signal stemming from the dominance of an HT conformer (DeltaHT in the adduct with 3'-GMPs and LambdaHT in the adduct with 5'-GMPs).

Topics: Antineoplastic Agents; Circular Dichroism; Cisplatin; Crystallography, X-Ray; DNA Adducts; Guanosine; Guanosine Monophosphate; Ligands; Models, Molecular; Molecular Structure; Nucleotides; Organoplatinum Compounds; Solvents; Temperature

Proteins interact with nucleotides to perform a multitude of functions within cells. These interactions are highly specific; however, the molecular basis for this specificity is not well understood. To identify factors critical for protein-guanine nucleotide recognition the binding of two closely related ligands, guanosine 3'-monophosphate (3'GMP) and inosine 3'-monophosphate (3'IMP), to Ribonuclease Sa (RNase Sa), a small, guanylyl-endoribonuclease from Streptomyces aureofaciens, was compared using isothermal titration calorimetry, NMR, X-ray crystallography and molecular dynamics simulations. This comparison has allowed for the determination of the contribution of the exocyclic amino group "N2" of the guanine base to nucleotide binding specificity. Calorimetric measurements indicate that RNase Sa has a higher affinity for 3'GMP (K=(1.5+/-0.2)x10(5)) over 3'IMP (K=(3.1+/-0.2)x10(4)) emphasizing the importance of N2 as a key determinant of RNase Sa guanine binding specificity. This result was unexpected as the published structural data for RNase Sa in complex with 3'GMP showed only a potential long-range interaction (>3.3A) between N2 and the side-chain of Glu41 of RNase Sa. The observed difference in affinity is largely due to a reduction in the favorable enthalpy change by 10 kJ/mol for 3'IMP binding as compared to 3'GMP that is accompanied by a significant difference in the heat capacity changes observed for binding the two ligands. To aid interpretation of the calorimetric data, the first crystal structure of a small, guanylyl ribonuclease bound to 3'IMP was determined to 2.0 A resolution. This structure has revealed small yet unexpected changes in the ligand conformation and differences in the conformations of the side-chains contacting the sugar and phosphate moieties as compared to the 3'GMP complex. The structural data suggest the less favorable enthalpy change is due to an overall lengthening of the contacts between RNase Sa and 3'IMP as compared to 3'GMP. The long-range interaction between N2 and Glu41 is critical for positioning of the nucleotide in the binding cleft for optimal contact formation. Thus, combined, the data demonstrate how a long-range interaction can have a significant impact on nucleotide binding affinity and energetics.

Topics: Binding Sites; Crystallography, X-Ray; Guanosine Monophosphate; Inosine Monophosphate; Isoenzymes; Molecular Conformation; Nucleotides; Protein Binding; Ribonucleases; Streptomyces aureofaciens; Substrate Specificity; Thermodynamics

The backbone dynamics of RNase T1 in the presence of exo-guanosine 2',3'-cyclophosphorothioate (exo-cGPS isomer), which is a productive substrate, and in the presence of 3'-guanylic acid (3'GMP), which is an nonproductive substrate, were examined using (15)N nuclear magnetic resonance. Although the X-ray crystal structure suggests that the modes of binding of these substrates to the active-site cleft are very similar, the order parameters in a number of regions in RNase T1 complexed with exo-cGPS isomer were different from those with 3'GMP. Moreover, the chemical exchange in line width observed for RNase T1 complexed with exo-cGPS isomer was also different from that observed for RNase T1 complexed with 3'GMP. From these results, we concluded that the internal motions in RNase T1 complexed with a productive substrate were not always identical to those in RNase T1 complexed with a nonproductive substrate.

Topics: Cyclic GMP; Guanosine Monophosphate; Models, Molecular; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Ribonuclease T1; Thionucleotides

Using the binding of a nucleotide inhibitor (guanosine-3'-monophosphate) to a ribonuclease (ribonuclease Sa) as a model system, we show that the salt-dependence of the interaction arises due to specific ion binding at the site of nucleotide binding. The presence of specific ion-protein binding is concluded from a combination of differential scanning calorimetry and NMR data. Isothermal titration calorimetry data are then fit to determine the energetic profile (enthalpy, entropy, and heat capacity) for both the ion-protein and nucleotide-protein interactions. The results provide insight into the energetics of charge-charge interactions, and have implications for the interpretation of an observed salt-dependence. Further, the presence of specific ion-binding leads to a system behavior as a function of temperature that is drastically different from that predicted from Poisson-Boltzmann calculations.

Topics: Binding Sites; Binding, Competitive; Calorimetry, Differential Scanning; Enzyme Stability; Escherichia coli; Guanosine Monophosphate; Ions; Isoenzymes; Ligands; Models, Chemical; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Proteins; Recombinant Proteins; Ribonucleases; Salts; Thermodynamics

Rate constants for guanine rotation about the Pt-N7 bond in (R,S,S,R)-Me(2)DABPtG(2) complexes (Me(2)DAB = N,N'-dimethyl-2,3-diaminobutane; G = 9-EtG, 3'-GMP, and 5'-GMP) were evaluated from line-shape analysis of H8 resonances. Three diastereomers, two in head-to-tail (DeltaHT and LambdaHT) and one in head-to-head (HH) conformations, exist in equilibrium in solution. The two guanines are equivalent in DeltaHT and LambdaHT conformers and nonequivalent in the HH form; therefore, four rate constants (k(Delta)(HT), k(Lambda)(HT), k(HH)()s, and k(HH)()d; sub-subscripts s and d stand for H8-shielded and -deshielded guanine, respectively) were evaluated. Activation parameters (DeltaH and DeltaS) were evaluated from the rate constant dependence on temperature. High values of DeltaH (78-93 kJ mol(-)(1)) and DeltaS (51-71 J K(-)(1) mol(-)(1)) were found for G rotation in the preferred DeltaHT rotamer having the six-membered ring of each guanine more canted toward the cis-G and a favorable dipole-dipole internucleotide interaction. Lower values of DeltaH() (64-76 kJ mol(-)(1)) and very small values of DeltaS() (-7-11 J K(-)(1) mol(-)(1)) were found for G rotation in the less favorable LambdaHT rotamer, indicating that the ground-state entropy of this rotamer is close to that of the activated complex and the ground-state enthalpy closer to that of the activated complex than for the DeltaHT rotamer. For the two guanines in the HH rotamer there is no large difference in activation parameters. In general DeltaH falls in the range 66-84 kJ mol(-)(1) (rather close to the values for the LambdaHT rotamer) and DeltaS in the range 14-41 J K(-)(1) mol(-)(1). The equilibrium constant between HT and HH rotamers was also evaluated together with the corresponding thermodynamic parameters (DeltaH and DeltaS). It is found that the low enthalpy is the major stabilizing factor for DeltaHT as compared to HH, while the entropy factor would favor the latter rotamer. In contrast the greater entropy is the stabilizing factor for the LambdaHT rotamer (the second most abundant conformer for 9-EtG and 3'-GMP) over the HH rotamer. In the latter case the enthalpy would favor the HH rotamer. In the case of the 5'-GMP derivative the greater entropy of the LambdaHT rotamer is not such to compensate for the lower enthalpy of the HH rotamer, and the latter remains the second most abundant rotamer. This investigation has allowed for the first time the enthalpic and entropic contributions favoring di

Topics: Chemical Phenomena; Chemistry, Physical; Guanine; Guanosine Monophosphate; Kinetics; Ligands; Models, Molecular; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Stereoisomerism; Thermodynamics

Ribonuclease from Streptomyces aureofaciens, the bacterial source for the industrial production of chlorotetracycline, is a guanylate endoribonuclease (RNase Sa; EC 3.1.27.3) which hydrolyses the phosphodiester bonds of single-stranded RNA at the 3'-side of guanosine nucleotides with high specificity. The structure of the enzyme was previously refined at atomic resolution (1.2 A) using room-temperature data. Here, the RNase Sa structure refined against 1.0 A data collected at cryogenic temperature is reported. There are two surface loops in molecule A and one in molecule B for which two main-chain conformations are modelled: these loops contain active-site residues. The separation for most of the corresponding main-chain atoms in the two conformations is about 0.8 A, with a maximum of 2.5 A. The two regions of dual conformation represent the most important differences in comparison with the structure determined at room temperature, where the corresponding loops have one conformation only but the largest degree of anisotropy. The flexibility of the loops observed in the structure of RNase Sa is directly linked to the need for the active site to interact productively with substrates and/or inhibitors.

Topics: Catalytic Domain; Crystallography, X-Ray; Guanosine Monophosphate; Isoenzymes; Models, Molecular; Molecular Structure; Protein Conformation; Ribonucleases; Static Electricity; Streptomyces aureofaciens; Temperature

Drug discovery procedures based on NMR typically require the analysis of thousands of NMR spectra. For example, in "SAR by NMR", two-dimensional NMR spectra are recorded for a target protein mixed with ligand candidates from a comprehensive library of small molecules and are compared to the corresponding spectrum for the protein alone. We present an automated procedure for the comparative analysis of large sets of heteronuclear single quantum coherence spectra, which is based on three-way decomposition and implemented as the software package MUNIN. In a single step, spectra with differences in the peak positions (indicating ligand binding) and the affected peaks are identified. By omission of peak picking, ad hoc scoring of the quality of doubtful peaks is avoided. The procedure has been tested on the bacterial ribonuclease barnase, with a protein concentration of only 50 microM, using several small molecules including the substrate analogue 3'-GMP. Sets of 51 spectra were processed simultaneously, and it is concluded that spectra with binding ligands can be unambiguously identified from much larger sets of spectra.

Topics: Bacterial Proteins; Drug Design; Guanosine Monophosphate; Ligands; Magnetic Resonance Spectroscopy; Nitrogen Isotopes; Quantitative Structure-Activity Relationship; Ribonucleases

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.

Topics: Aspergillus oryzae; Binding Sites; Catalysis; Crystallography, X-Ray; Deoxyuridine; Dinucleoside Phosphates; Guanosine Monophosphate; Ligands; Macromolecular Substances; Models, Molecular; Organophosphonates; Ribonuclease T1; Stereoisomerism; Substrate Specificity; Thermodynamics

During the growth cycle of the protozoan parasite Crithidia luciliae, there was a dramatic concomitant increase in the rate of adenosine and guanosine transport and 3' nucleotidase (3'NTase) activity after 72-94 hr. The simultaneous increased activities of the nucleoside transporters and 3'NTase could be suppressed by addition to the medium of a purine supplement such as adenosine (100 microM). C. luciliae grown in purine-replete medium (> or = 75 microM adenosine) exhibited low rates of adenosine and guanosine transport whilst parasites transferred to a defined serum-free medium containing < or = 7.5 microM adenosine demonstrated elevated levels of both adenosine and guanosine transport up to 25- to 40-fold. The increased activity of the nucleoside transporters was inhibited by cycloheximide (10 microM). Under conditions of purine depletion 3'AMP and 3'GMP inhibited the adenosine and guanosine transporters, respectively. However, in the presence of a purine supplement (100 microM), neither 3'AMP nor 3'GMP was an effective inhibitor of nucleoside transport. Our results link the increased activity of the nucleoside transporters to the increased activity of the 3'NTase, indicating the activation of a purine salvage system not previously reported in other organisms.

Topics: Adenosine; Adenosine Monophosphate; Animals; Biological Transport; Carrier Proteins; Crithidia; Cycloheximide; Guanosine; Guanosine Monophosphate; Hydrogen-Ion Concentration; Membrane Proteins; Nucleoside Transport Proteins; Nucleotidases; Protein Synthesis Inhibitors; Purine Nucleosides

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)

Topics: Catalysis; Computer Simulation; Energy Metabolism; Guanosine Monophosphate; Hydrogen-Ion Concentration; Models, Theoretical; Ribonuclease T1

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.

Topics: Binding Sites; Crystallography, X-Ray; Glutamates; Glutamic Acid; Guanosine Monophosphate; Histidine; Hydrogen Bonding; Hydroxyl Radical; Isomerism; Models, Molecular; Molecular Sequence Data; Protein Conformation; Ribonuclease T1

A modified method for the synthesis and separation of endo and exo guanosine 2',3'-cyclophosphorothioate (cGPS) has been developed. The exo diastereoisomer has been co-crystallized with RNase T1. cGPS is known to be a RNase T1 inhibitor but is also a very slow substrate. It was hydrolyzed during the crystallization, leaving 3'-guanylic acid (3'-GMP) in the active site. As a guanosine was also found to be bound in a subsite, the enzyme contains the products of the reaction of guanylyl-3',5'-guanosine. The structure was refined to a resolution of 1.7 A and yielded a final R value of 14.5%. In contrast to previous 3'-GMP complexes of RNase T1, the ribose phosphate moiety of the inhibitor is in contact with all the active site residues. The phosphate forms hydrogen bonds with Asn36, Tyr38, Arg77, His92, and with Asn49 from a symmetry-related molecule. The ribose 2'-OH is hydrogen-bonded to both Glu58 and His40. The interactions in the active site of the present structure are compared to those found in the 2'-GMP complex of RNase T1.

Topics: Binding Sites; Crystallography, X-Ray; Cyclic GMP; Guanosine; Guanosine Monophosphate; Hydrolysis; Molecular Structure; Ribonuclease T1; Stereoisomerism; Thionucleotides

We have theoretically and experimentally studied the binding of two different ligands to wild-type ribonuclease T1 (RNT1) and to a mutant of RNT1 with Glu-46 replaced by Gln. The binding of the natural substrate 3'-GMP has been compared with the binding of a fluorescent probe, 2-aminopurine 3'-monophosphate (2AP), and relative free energies of binding of these ligands to the mutant and the wild-type (wt) enzyme have been calculated by free energy perturbation methods. The free energy perturbations predict that the mutant RNT1-Gln-46 binds 2AP better than 3'GMP, in agreement with experiments on dinucleotides. Four free energy perturbations, forming a closed loop, have been performed to allow the detection of systematic errors in the simulation procedure. Because of the larger number of atoms involved, it was necessary to use a much longer simulation time for the change in the protein, i,e., the perturbation from Glu to Gln, than in the perturbation from 3'-GMP to 2AP. Finally the structure of the binding site is analyzed for understanding differences in catalytic speed and binding strength.

Topics: 2-Aminopurine; Computer Simulation; Dinucleoside Phosphates; Guanosine Monophosphate; Ligands; Models, Chemical; Models, Molecular; Point Mutation; Ribonuclease T1; Substrate Specificity; Thermodynamics

The crystal structure of the complex between ribonuclease T1 and 3'GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 N epsilon H+ activates O2'-H, (d) Glu58 carboxylate acts as base and His92 N epsilon H+ as acid in a general acid-base catalysis. The crystals have the monoclinic space group P2(1), a = 4.968 nm, b = 4.833 nm, c = 4.048 nm, beta = 90.62 degrees with two molecules in the asymmetric unit. The structure was determined by molecular replacement and refined to R = 15.3% with 11,338 data > or = 1 sigma (Fo) in the resolution range 1.0-0.2 nm; this includes 180 water molecules and two Ca2+. The structure of ribonuclease T1 is as previously observed. 3'GMP is bound in syn conformation; guanine is located in the specific recognition site, the ribose adopts C4'-exo puckering, the ribose phosphate is extended with torsion angle epsilon in trans. The O2'-H group is activated by accepting and donating hydrogen bonds from His40 N epsilon H+ and to Glu58 O epsilon 1; the phosphate is hydrogen bonded to Glu58 O epsilon 2H, Arg77 N epsilon H+ and N eta 2H+, Tyr38 O eta H, His92 N eta H+. The conformation of ribose phosphate is such that O2' is at a distance of 0.31 nm from phosphorus, and opposite the P-OP3 bond which accepts a hydrogen bond from His92 N epsilon H+; we infer from a model building study that this bond is equivalent to the scissile P-O5' in a substrate GpN.

Topics: Binding Sites; Calcium; Chromatography, High Pressure Liquid; Crystallography, X-Ray; Guanine; Guanosine Monophosphate; Hydrogen Bonding; Isomerism; Models, Molecular; Molecular Conformation; Recombinant Proteins; Ribonuclease T1; Substrate Specificity

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.

Topics: Binding Sites; Computer Simulation; Guanosine Monophosphate; Kinetics; Models, Molecular; Ribonuclease T1; Spectrometry, Fluorescence; Thermodynamics

2',3'-cyclic nucleotides are intermediates and substrates of Ribonuclease (RNase)-catalysed reactions. The characterization of the equilibrium conformation as well as the flexibility inherent in these molecules helps in understanding the enzymatic action of RNases. The present study explores parameters like phase angle, glycosydic torsion angle and hydrogen bond to find possible interrelationship between them through Molecular Dynamics (MD) simulations on 3'-GMP,3'-UMP, A greater than p, G greater than p, U greater than p, C greater than p, GpA greater than p and UpA greater than p. Interesting results of the effect of cyclisation and other constraints such as hydrogen bond between certain groups on the equilibrium ribose conformation have emerged from this study.

Topics: Catalysis; Guanosine Monophosphate; Nucleic Acid Conformation; Nucleotides, Cyclic; Ribonucleases

Temperature-jump relaxation kinetic studies were undertaken at 25 degrees C with ribonuclease T1 (RNase T1) alone and in the presence of guanosine (Guo) and 3'-guanylic acid (3'-GMP). No relaxations were observed in the absence of ligands and only one process was observed in their presence which reflected a simple on-off reaction in both cases. Apparent association rate constants, k(on), and dissociation rate constants, k(off), were evaluated at several pH values and their ratios, k(on)/k(off), were contrasted with independently determined values of the equilibrium association constant, Ka(eq). The value of k(on)/k(off) for Guo was significantly greater than Ka(eq), whereas Ka(eq) was significantly greater than k(on)/k(off) for 3'-GMP. The simplest interpretation of the result for Guo is that free RNase T1 undergoes a relatively slow undetected isomerization and Guo can bind only with one isomer. 3'-GMP can be considered to bind with the same preference, but in this case the initial enzyme complex undergoes a relatively slow undetected isomerization. These results are consistent with a recent NMR study which suggested that RNase T1 binding with Guo and 3'-GMP are coupled to slow exchange processes in a ligand dependent manner (Shimada, I. and Inagaki, F. (1990) Biochemistry 29, 757-764). It is tentatively concluded that binding of Guo and 3'-GMP at the active site of RNase T1 is limited to a sub-population of conformers involving the base-recognition site and that the phosphomonoester group of the nucleotide can engage in additional conformationally linked interactions at the adjacent catalytic site.

Topics: Guanosine; Guanosine Monophosphate; Kinetics; Protein Conformation; Ribonuclease T1

The crystal structures of ribonuclease from Streptomyces aureofaciens (RNase Sa) and its complex with 3'-guanylic acid (guanosine 3'-monophosphate, 3'-GMP) have been determined by the method of isomorphous replacement. The atomic parameters have been refined by restrained least-squares minimization using data in the resolution range 10.0-1.8 A. All protein atoms and more than 230 water atoms in the two crystal structures have been refined to crystallographic R factors of 0.172 and 0.175 respectively. The estimated r.m.s. error in the atomic positions ranges from 0.2 A for well-defined atoms to about 0.5 A for more poorly defined atoms. There are two enzyme molecules in the asymmetric unit, built independently, and referred to as molecules A and B. The value of the average B factor for protein atoms in both structures is about 19 A2 and for water molecules about 35 A2. Electron density for the substrate analogue 3'-GMP was found only at the active site of molecule A. The density was very clear and the positions of all 3'-GMP atoms were refined with precision comparable to that of the protein.

Topics: Amino Acid Sequence; Binding Sites; Chemical Phenomena; Chemistry, Physical; Crystallization; Guanosine Monophosphate; Hydrogen Bonding; Molecular Sequence Data; Molecular Structure; Protein Conformation; Ribonucleases; Streptomyces aureofaciens; X-Ray Diffraction

Hydrophobic effects on binding of ribonuclease T1 to guanine bases of several ribonucleotides have been proved by mutating a hydrophobic residue at the recognition site and by measuring the effect on binding. Mutation of a hydrophobic surface residue to a more hydrophobic residue (Tyr45----Trp) enhances the binding to ribonucleotides, including mononucleotide inhibitor and product, and a synthetic substrate-analog trinucleotide as well as the binding to dinucleotide substrates and RNA. Enhancements on binding to non-substrate ribonucleotides by the mutation have been observed with free energy changes ranging from -2.2 to -3.9 kJ/mol. These changes are in good agreement with that of substrate binding, -2.3 kJ/mol, which is calculated from Michaelis constants obtained from kinetic studies. It is shown, by comparing the observed and calculated changes in binding free energy with differences in the observed transfer free energy changes of the amino acid side chains from organic solvents to water, that the enhancement observed on guanine binding comes from the difference in the hydrophobic effects of the side chains of tyrosine and tryptophan. Furthermore, a linear relationship between nucleolytic activities and hydrophobicity of the residues (Ala, Phe, Tyr, Trp) at position 45 is observed. The mutation could not change substantially the base specificity of RNase T1, which exhibits a prime requirement for guanine bases of substrates.

Topics: Binding Sites; Guanosine Monophosphate; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Oligoribonucleotides; Protein Conformation; Ribonuclease T1; Ribonucleotides; RNA; Surface Properties; Tryptophan

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.

Topics: Binding Sites; Chemical Phenomena; Chemistry, Physical; Diffusion; Guanosine Monophosphate; Hydrogen-Ion Concentration; Protein Conformation; Ribonuclease T1; Spectrometry, Fluorescence; Temperature; Thermodynamics