Electrostatic potential of nucleotide-free protein is sufficient for discrimination between adenine and guanine-specific binding sites

Despite sharing many common features, adenine-binding and guanine-binding sites in proteins often show a clear preference for the cognate over the non-cognate ligand. We have analyzed electrostatic potential (ESP) patterns at adenine and guanine-binding sites of a large number of non-redundant proteins where each binding site was first annotated as adenine/guanine-specific or non-specific from a survey of primary literature. We show that more than 90% of ESP variance at the binding sites is accounted for by only two principal component ESP vectors, each aligned to molecular dipoles of adenine and guanine. Projected on these principal component vectors, the adenine/guanine-specific and non-specific binding sites, including adenine-containing dinucleotides, show non-overlapping distributions. Adenine or guanine specificities of the binding sites also show high correlation with the corresponding electrostatic replacement (cognate by non-cognate ligand) energies. High correlation coefficients (0.94 for 35 adenine-binding sites and 1.0 for 20 guanine-binding sites) were obtained when adenine/guanine specificities were predicted using the replacement energies. Our results demonstrate that ligand-free protein ESP is an excellent indicator for discrimination between adenine and guanine-specific binding sites and that ESP of ligand-free protein can be used as a tool to annotate known and putative purine-binding sites in proteins as adenine or guanine-specific.     

Purification and characterization of a human urine ribonuclease (RNAase 1) showing genetic polymorphism

A ribonuclease (RNAase) was isolated and purified from the urine of a 45-year-old man by column chromatographies on DEAE-Sepharose CL-6B, cellulose phosphate and CM-cellulose followed by gel filtrations on Bio-Gel P-100 and Sephadex G-75, and finally to a homogeneous state by SDS-polyacrylamide gel electrophoresis. The enzyme was designated RNAase 1. It was possible to detect RNAase 1 isozymes in urine and serum without difficulty using isoelectric focusing electrophoresis followed by immunoblotting with a rabbit antibody specific to RNAase 1. The existence of genetic polymorphism of RNAase 1 was detected in human serum utilizing this technique (Yasuda, T. et al. (1988) Am. J. Hum. Genet., in press). RNAase 1 in serum and urine seemed to exist in multiple forms with regard to molecular weight and pI value. Genetically polymorphic RNAase 1 was a glycoprotein, containing three mannose, one fucose, four glucosamine and no sialic acid residues per molecule, with a molecular weight of 16,000 and 17,500 determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme was most active at pH 7.0 on yeast RNA substrate and inhibited remarkably by Cu2+, Hg2+ and Zn2+. It also showed definite substrate preference for poly(C) and poly(U), but much less activity against poly(A) and poly(G). Thus, the enzyme is a pyrimidine-specific RNAase.     

Distribution of the inhibiting activity among bivalent metal cations

The inhibition coefficients (beta) of bovine pyrimidine-specific RNAase A by bivalent metal cations were determined. The effect of ionization potentials, hydration energy and ionic radii of cations on their inhibiting activity is discussed.     

Recognition pattern of different bases in the active site of ribonuclease Ms--a model building study

The structure of base non-specific ribonuclease Ms from Aspergillus saitoi was predicted by sequence similarity to guanine-specific RNase T1 of known structure. In this paper the interaction pattern of binding site of RNase Ms with different nucleic acids bases is analysed using model building and energy minimisation techniques. It is shown that unspecificity of this protein can be explained only when taking into account flexibility of the base recognition loop.     

Extracellular RNAase Pch2 of Penicillium chrysogenum 152A: purification, specificity and physico-chemical properties

Acid RNAase Pch2 was isolated from a filtrate of the cultural fluid of the fungus Penicillium chrysogenum 152A and purified to homogeneity. An analysis of RNAase Pch2 action on RNA and synthetic substrates showed that the enzyme can be attributed to non-specific true ribonucleases (ribonucleate-3'-oligo-nucleotide hydrolase, EC 3.1.4.23). The maximal effect of the enzyme on RNA is observe at pH 4.5 and 55 degree. The RNAase Pch2 is not activated by bivalent metal ions, p-chloromercurybenzoate or beta-mercaptoethanol and is reversibly inactivated by 8 M urea. The enzyme molecule consists of 332 amino acid residues; its molecular weight is 36160, the isoelectric point lies at 5.2.     

Jon Widom,the Scientist

Abstract

The structure of base non-specific ribonuclease Ms from Aspergillus saitoi was predicted by sequence similarity to guanine-specific RNase T1 of known structure. In this paper the interaction pattern of binding site of RNase Ms with differnt nucleic acids bases is analysed using model building and energy minimisation techniques. It is shown that unspecificity of this protein can be explained only when taking into account flexibility of the base recognition loop.     

The properties of the interaction between bovine pancreatic ribonuclease a and mononucleotides supports the existence of several binding sub-sites in the enzyme

The binding of 5'AMP, 5'GMP, 5'CMP, 3'CMP and Cl6RMP to RNAase A was studied by means of the gel filtration technique. It was found that only one molecule of 3'CMP binds strongly to the enzyme although a very unspecific binding is also present. The interaction of 5'AMP and 5'GMP with the enzyme shows one strong binding site and several weak binding sites, whereas two molecules of 5'CMP bind to RNAase A with equal strength. Cl6RMP shows an anomalous behaviour as both split peaks and troughs are found in the chromatogram. The Ka values for 3'CMP and the strong binding site of 5'AMP and 5'GMP are very similar whereas that for the two binding sites of 5'CMP is smaller (about 2.2 X 10(-4)M-1 and 0.5 X 10(-4)M-1, respectively at pH 5.5, I = 0.01 and 25 degrees C). The results are in general agreement with the known multiplicity of ligand-binding subsites in RNAase A.     

1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A

The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5'AMP, 5'GMP, 5'IMP) and halogenated purine mononucleotides (cl6RMP, br8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in the pK values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p1 site, but the p2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571--579). Lys-7 and/or Arg-10 are proposed as part of the p2 phosphate-binding subsite. The pK values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5'AMP, 5'GMP and 5'IMP takes place not only in the so-called purine-binding site B2R2p1 but also in the primary pyrimidine-binding site B1R1 and p0 of RNAase A.     

A rapid method for the isolation of ribonuclease from yeast (Saccharomyces carlsbergensis).

A ribonuclease (RNAase; EC 3.1.14.1) from brewer's yeast was purified 90-fold. Crude RNAase was initially separated from other proteins by precipitation at pH 4.0 after incubation of the mechanically disrupted yeast cells at pH 6.0 and 52 degrees C for 30 min. The RNAase was purified from the supernatant by ultrafiltration with a PM-30 membrane and adsorption chromatography on hydroxyapatite. RNAase preparation was free of phosphatase, deoxyribonuclease and phosphodiesterase activities. It showed maximum activity at pH 6.0 and a temperature optimum of 52 degrees C with yeast RNA as substrate. This RNAase hydrolysed yeast RNA to nucleoside 3'-phosphates and showed no evidence of base specificity.     

Purification and properties of two ribonucleases and a nuclease from barley seeds

Three enzymes possessing RNAase activity were isolated from barley seeds. These enzymes were further purified by ammonium sulphate precipitation DEAE-cellulose chromatography, gel filtration on Sephadex G-75 and DEAE-Sephadex A-50 chromatography. These enzymes have been characterized and classified as: 1. Plant RNAase I (EC 3.1.27.1). It has a pH optimum at 5.7 and molecular weight of 19 000. 2. Plant RNAase II (EC 3.1.27.1). It has a pH optimum at 6.35 and molecular weight of 19 000. 3. Plant nuclease I (EC 3.1.30.2). It has a pH optimum at 6.8 and molecular weight of 37 000. Two RNAases were purified to homogeneity by means of affinity chromatography on poly(G)-Sepharose 4B, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Pharmacokinetic properties of pancreatic and microbial ribonucleases

Pharmacokinetic properties of pancreatic RNAase (RNAase I), RNAase of Bacillus intermedius (RNAase Bi) and RNAase of Streptomyces rimosus (RNAase Sr) were studied on albino rats. RNAase Bi was shown to be characterized by a higher rate and level of absorption into the systemic blood flow, higher retention time, lower elimination from the kidneys and tissues of the peripheral chamber (skeletal muscles) and higher distribution in the other animal organs such as the heart, spleen and brain. It was concluded by the experimental results that the higher antiviral efficacy of RNAase Bi (RNAase Bi greater than RNAase Sr greater than RNAase I), as was known from the literature data, and the ability to stimulate the immunity correlated with higher biological availability of the enzyme in the animals and could be due to its pharmacokinetic properties.     

Reversible inactivation of ribonucleases by ADPribosylation

The activity of purified bovine seminal RNAase and pancreatic RNAase A (EC 3.1.27.5) has been investigated following in vitro ADPribosylation in the presence of nuclear ADPribosyltransferase (EC 2.4.2.30) and NAD+ X ADPribosylation of these enzymes was correlated with a significant decrease in their activities. Approximately three residues of ADPribose were present per mol of enzyme. Removal of the bound ADPribose restored enzyme activity to near normal levels. Similar results were obtained with nuclei isolated from bull seminal vesicles as an endogenous source of seminal RNAase and nuclear ADPribosyltransferase. The findings suggest that in vitro ADPribosylation has a reversible inactivating effect on ribonucleases.     

The number and role of histidine residues in the active site of guanyloribonuclease Sa

The number and role of histidine residues in the active site of extracellular guanyloribonuclease Sa produced by Streptomyces aureofaciens (RNAase Sa) were studied via chemical modification by ethoxyformic anhydride by means of circular dichroism measurements. It was shown that only one of two histidines of RNAase Sa is situated in the active site of the enzyme. Ethoxyformylation of RNAase Sa in the presence of Guo-3'-P, Guo-5'-P and dGuo-5-P, all of them being competitive inhibitors of the enzyme, supported the assumption that an essential histidine residue is bound to the phosphate group in the position 3' of the ribose ring. The circular dichroism measurements of native and modified RNAase Sa and of its complex with Guo-3'-P showed that the modification of the essential histidine residue resulted in alteration of binding of RNAase Sa to Guo-3'-P; histidine thus may play a key role in the formation of such a complex.     

Pyrimidine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus--biochemical characterization and homology modeling

We report the characterization of the pyrimidine-specific ribonucleoside hydrolase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsCU-NH). The gene SSO0505 encoding SsCU-NH was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. SsCU-NH is a homotetramer of 140 kDa that recognizes uridine and cytidine as substrates. SsCU-NH shares 34% sequence identity with pyrimidine-specific nucleoside hydrolase from E. coli YeiK. The alignment of the amino acid sequences of SsCU-NH with nucleoside hydrolases whose 3D structures have been solved indicates that the amino acid residues involved in the calcium- and ribose-binding sites are preserved. SsCU-NH is highly thermophilic with an optimum temperature of 100 degrees C and is characterized by extreme thermodynamic stability (T(m) = 106 degrees C) and kinetic stability (100% residual activity after 1 h incubation at 90 degrees C). Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. The structure of the enzyme determined by homology modeling provides insight into the proteolytic analyses as well as into mechanisms of thermal stability. This is the first nucleoside hydrolase from Archaea.     

Identification of ribonuclease P activity from chick embryos

RNAase P (EC 3.1.26.5) activity has been identified in chick embryo thigh tissue on the basis of specific cleavage of Escherichia coli 129 nucleotide tRNATyr precursor and has been partially purified by the procedure used for human tissue culture KB cell RNAase P. RNAase P from chick resembles the KB cell RNAase P in substrate specificity, requirement for a divalent cation (Mg2+) and a monovalent cation (K+, Na+ or NH4+) for activity, inhibition by bulk tRNA, ready inactivation by proteases, and increasing instability; with purification. RNAase P activity is also present in whole chick embryos, as well as in liver and heart tissues. Furthermore, crude preparations of RNAase P from chick embryo heart tissue are relatively free of contaminating nucleases.     

Investigation of binding sites in the complex pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa

The pyr-3 gene of Neurospora crassa codes for the bifunctional enzyme pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase (carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating)/carbamoylphosphate: L-aspartate carbamoyltransferase), EC 6.3.4.16/EC 2.1.3.2). We describe the investigation of substrate- and product-binding sites of the enzyme by affinity chromatography, using the ligands aspartate, glutamate, and adenosine 5'-diphosphate, and investigate the channelling of carbamoyl phosphate, the product of the first function and substrate of the second, through the pathway. For this latter aspect of the investigation, two new enzyme assays were devised and described. The results of the competition studies on carbamoyl phosphate-binding are consistent with the existence of two different binding sites within the enzyme for this metabolic intermediate, one for it as the product of the first step and the other for it as the substrate of the second.     

Puridication and properties of an intracellular ribonuclease from Candida lipolytica.

1. A ribonuclease (RNAase CL) (EC 3.1.4.23, ribonucleate 3'-oligonucleotide hydrolase) was extracted by EDTA/acetate buffer, pH 5.6 from acetonedried cells of Candida lipolytica and purified 1350-fold by acetone and (NH4)2SO4 fractionation, DEAE-cellulose and DEAE-Sephadex chromatography. 2. RNAase CL is an acidic protein having an isoelectric point of 4.2, and an approximate molecular weight of 32 000. 3. Optimal pH and temperature for the enzyme were 6.0 and 60 degrees C, respectively. It is stable at neutral pH up to 50 degrees C. At 64 degrees C for 30 min, 95, 49 and 64% inactivation of the enzyme occurred at pH values 4.2, 6.6 and 10.0, respectively. 4. RNAase CL inhibited by Zn2+ and Cu2+, sulfhydryl reactants and by high concentration of salts, but not by chelating agents. 5. RNAase CL degraded ribosomal RNA, transfer RNA, polyadenylic acid, polycytidylic acid and polyuridylic acid into acid-soluble nucleotides. Among the synthetic homopolymers, polycytidylic acid was most rapidly degraded. Polyguanylic acid and duplexes of synthetic homopolymers were less sensitive. DNA was not attacked. Specificity studies showed that RNAase CL preferentially cleaves pC-purine bonds. 6. Digestion of poly (C) by RNAase CL resulted in the liberation of cyclic 2',3'-CPM from the start of the reaction with no observable formation of intermediate oligonucleotides. This suggests that the enzyme depolymerizes by an exonucleolytic mechanism.     

High sequence specificity of micrococcal nuclease.

The substrate specificity of micrococcal nuclease (EC 3.1.4.7.) has been studied. The enzyme recognises features of nucleotide composition, nucleotide sequence and tertiary structure of DNA. Kinetic analysis indicates that the rate of cleavage is 30 times greater at the 5' side of A or T than at G or C. Digestion of end-labelled linear DNA molecules of known sequence revealed that only a limited number of sites are cut, generating a highly specific pattern of fragments. The frequency of cleavage at each site has been determined and it may reflect the poor base overlap in the 5' T-A 3' stack as well as the length of contiguous A and T residues. The same sequence preferences are found when DNA is assembled into nucleosomes. Deoxyribonuclease 1 (EC 3.1.4.5.) recognises many of the same sequence features. Micrococcal nuclease also mimics nuclease S1 selectively cleaving an inverted repeat in supercoiled pBR322. The value of micrococcal nuclease as a "non-specific" enzymatic probe for studying nucleosome phasing is questioned.