Novel extracellular ribonuclease from Bacillus intermedius--binase II: purification and some properties of the enzyme.

The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene. The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel. The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg. The molecular weight of binase II is 30 kD. The enzyme is activated by Mg2+ and virtually completely inhibited by EDTA. The pH optimum for the reaction of RNA hydrolysis is 8.5. The properties of the enzyme are close to those of RNase Bsn from B. subtilis. The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom.

The effect of net charge on the solubility, activity, and stability of ribonuclease Sa.

The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.

Contribution of arginine-82 and arginine-86 to catalysis of RNases from Bacillus intermedius (binase).

To elucidate the functional role of Arg82 and Arg86 in the enzyme activity of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant Arg86 Ala is 2.7 x 10(3) - 7.7 x 10(3) times less than that of the native enzyme. The decrease in activity is determined preferentially by the decrease in the molecular rate constant kcat with a relatively small change of enzyme-substrate affinity, characterized by Km. This is the expected result if Arg86 acts to lower the energy of a transition state of the reaction. The replacement of Arg82 by Ala causes a 5-19-fold activity decrease, depending on the substrate. We propose that this residue does not have a direct catalytic function in the molecular mechanism of the binase action and that the activity decrease of binase on the replacement of Arg82 by alanine is mediated by the effect of Arg82 on the pK of catalytic residues.

Determination of the nucleotide conformation in the productive enzyme-substrate complexes of RNA-depolymerases.

The aim of this work is to determine the conformation of the nucleobase adjacent to the cleavable phosphodiester bond in the productive enzyme-substrate complex of RNA-depolymerizing enzymes. To this end the kinetic parameters of hydrolysis of UpA, 2'-C-Me- and 3'-C-Me-UpA were determined for RNase A, RNase Pb2, nuclease S1 and snake venom phosphodiesterase. In these derivatives the ranges of the allowed orientation of uridine residues are restricted due to the substitution of methyl groups for the ribose hydrogen atoms. The results described demonstrate that the proposed method is of general value for the estimation of the nucleotide glycoside angles in the productive enzyme-substrate complexes.

A novel guanyl-preferable ribonuclease of Bacillus polymyxa: isolation and characterization of the enzyme.

Three forms of the extracellular alkaline ribonuclease of B. polymyxa (RNases Bpo I, Bpo II and Bpo III) were obtained in the homogeneous states. Relative molecular weights, N- and C-terminal amino acid sequences of the proteins were determined. The two higher-molecular weight forms of the enzyme (RNases Bpo I and Bpo III) were shown to be precursor molecules which differed from the mature B. polymyxa RNase (RNase Bpo II) by the presence of a extra twelve and six amino acid residues at the amino terminus, respectively. The study of catalytic properties of the enzyme elucidated that the extent of specificity of B. polymyxa RNase in respect to highpolymeric substrates is different from that of other natural Bacillus RNases. Amino acid composition of B. polymyxa RNase was established and structural similarity within family of Bacillus RNases is discussed. B. polymyxa RNase was shown to be inhibited by intracellular protein inhibitor of B. amyloliquefaciens RNase with the dissociation constant of 2.7 x 10(-12) M.

Dissociation constants and thermal stability of complexes of Bacillus intermedius RNase and the protein inhibitor of Bacillus amyloliquefaciens RNase.

Binase, the extracellular ribonuclease of Bacillus intermedius, is inhibited by barstar, the natural protein inhibitor of the homologous RNase, barnase, of B. intermedius. The dissociation constants of the binase complexes with barstar and its double Cys40,82Ala mutant are about 10(-12) M, only 5 to 43 times higher than those of the barnase-barstar complex. As with barnase, the denaturation temperature of binase is raised dramatically in the complex. Calorimetric studies of the formation and stability of the binase-barstar complex show that the binase reaction with barstar is qualitatively similar to that of barnase but some significant quantitative differences are reported.

Double-stranded RNA: the variables controlling its degradation by RNases.

The kinetics of single-stranded (SS) and double-stranded (ds) polyribonucleotides cleavage by three mammalian pancreatic type ribonucleases have been studied under low and high salt conditions. The values kcat, Km, and kcat/Km for depolymerization of poly(U), poly(A).poly(U), poly(I) and poly(I).poly(C) by bovine RNase A, bovine seminal RNase, and human seminal RNase have been determined and compared to each other. The Km values of bovine RNase A for (ss) or (ds) substrates were of the same order of magnitude under low and high ionic strength conditions, while their kcat values were found to differ considerably. Qualitatively similar results were obtained with bovine and human seminal RNases, i.e., the activity ratios (ssRNA/dsRNA) were mostly determined by the ratio of kcat values. It was shown that the modest levels of activity toward dsRNAs shown by single-strand-preferring RNases may occur by a mechanism consisting in the binding of the RNase to single nucleotides which are wound off the double helix because of thermal fluctuations. A higher activity and its enhancement as a function of number and location of the positive charges present on the RNase surface (human seminal RNase > bovine seminal RNase > bovine RNase A), as well as its increase under low ionic strength conditions, could instead be explained by the increased occurence of the splitting mechanism based on the binding of the RNase to single-stranded RNA sequences transiently exposed from the RNA double-helix.

Thermostability of the barnase-barstar complex.

Scanning microcalorimetry was used to study heat denaturation of barnase in complex with its intracellular inhibitor barstar. The heat denaturation of the barnase-barstar complex is well approximately by two two-state transitions with the lower temperature transition corresponding to barstar denaturation and the higher temperature one to barnase denaturation. The temperature of barnase melting in its complex with barstar is 20 degrees C higher than that of the free enzyme. The barstar melting temperature is almost the same in the complex or alone (71 degrees C at pH 6.2 and 68 degrees C at pH 8.0). It seems possible that when barstar unfolds it can remain bound to barnase, while the latter unfolds only on dissociation of the denatured barstar.

Mutational analysis of the active site of RNase of Bacillus intermedius (BINASE).

To elucidate the functional role of some residues in the active site of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant His101 Glu is 2.0-2.7% of that for the native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant kcat, with almost unchanged affinity of the enzyme for the substrate, characterized by KM. This is the expected result if His101 acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lys26 by Ala causes a reduction in the enzyme activity to 13-33%, depending on the substrate. The activity decreases are due to changes in both kcat and KM for poly(A) and poly(A) but in kcat alone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, barnase.

High sequence similarity between a ribonuclease from ginseng calluses and fungus-elicited proteins from parsley indicates that intracellular pathogenesis-related proteins are ribonucleases.

A ribonuclease from a callus cell culture of Panax ginseng C.A. Mey strain R1 was isolated. A pure protein with an apparent molecular mass of 18 kDa was obtained. The N-terminal sequences of the protein and of the C-terminal CNBr peptide were determined. No homology with other ribonucleases was found, but there was 60-70% sequence identity with two intracellular pathogenesis-related (IPR) proteins from parsley, indicating that not only these two proteins, but also homologous IPR proteins identified in other plant species are ribonucleases.

NMR studies of a complex of RNAse from Penicillium brevicompactum with dinucleoside phosphonate and the implications for the mechanism of enzyme action.

The chemical-shift dependences of the proton signals of the guanosine and uridine moieties were measured as a function of the relative amount of GpcU complexed with RNase Pb1 (EC 3.1.27.3). The equal values of the chemical-shift changes of the guanosine C8-protons on complex formation between GpcU and RNase Pb1 and that of the 3'-GMP and RNase Pb1 allow to conclude that the guanosine base is bound in the same manner in these protein-ligand complexes. The guanosine moiety of GpcU is also most probably bound in the syn-conformation. The absence of changes in both the linewidths and the chemical shifts of the C1', C5 and C6-proton signals of the uridine on complex formation indicates that the uridine moiety of the dinucleoside phosphonate is not immobilized in the complex. The pH dependences of the chemical shifts of the C2-protons of the histidine-imidazole ring of RNase Pb1 and that of the 31P of GpcU in the RNase complex were studied. The results suggest that there is a direct interaction between the phosphonate group of the ligand and the protonated imidazole ring of His-90. The side groups of His-38 and Glu-56 are hydrogen bonded to each other at neutral pH and they are located in the vicinity of the phosphonate group of GpcU. When the carboxyl group of Glu-56 is protonated the His-38 imidazole ring forms a new hydrogen bond with one of the phosphoryl oxygens of the phosphonate group. On the basis of these results we propose the mechanism of action of RNase Pb1 which is probably also true for RNase T1.

Increase of specificity of RNase from Bacillus amyloliquefaciens (barnase) by substitution of Glu for Ser57 using site-directed mutagenesis.

Bacterial ribonucleases from Bacillus amyloliquefaciens and Bacillus intermedius show the specificity towards the nature of a nucleoside at the O3' end of the phosphodiester bond to be split in the preference order G > A >> U > C in the cleavage reactions of polynucleotides. It follows from the X-ray data that the substrate guanosine base is bound at the active site of these RNases in the same manner as for high-specificity guanylic RNases. We supposed that the difference in specificity for the two types of RNases is due to the additional hydrogen bond between the protein and a purine base in the case of bacterial guanyl-preferring RNases in contrast to the high-specificity guanylic RNases. To examine this hypothesis we prepared the Ser57 --> Glu mutant of B. amyloliquefaciens, in which this hydrogen bond is eliminated. Kinetic studies demonstrate that the specificity of this mutant towards guanylic substrates is 35-times greater than that of the wild-type RNases from B. amyloliquefaciens and close to that of RNases T1.

A comparative study on the catalytic properties of guanyl-specific ribonucleases.

The kinetic parameters of reactions catalyzed by four guanyl-specific RNases T1, Pb1, Th1 and Sa were studied comparatively using three types of substrates; guanosine-2',3'-cyclophosphates, GpN dinucleoside phosphates and synthetic polyribonucleotides. The kinetic parameters were shown to be similar in spite of considerable differences in primary structures of these RNases, including amino acid residues of the active sites. Therefore, primary structures of guanyl RNases allow for a considerable number of substitutions (both in the 'recognising' and catalytical parts of the active site) without changes in the catalytical parameters.

A route to 2',5'-oligoadenylates with increased stability towards phosphodiesterases.

The rates of enzymatic hydrolysis of 2',5'-oligoadenylates and their synthetic analogs have been measured. These compounds were treated with either NIH 3T3 cell lysates, mouse liver homogenates or snake venom phosphodiesterase. All analogs with 3'-terminal acyclic nucleoside residues demonstrated greater stability compared with the natural compound adenylyl(2'-5')adenylyl(2'-5')adenosine.

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease.

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).

Stereoelectronic effects in RNase-catalysed reactions of dinucleoside phosphate cleavage.

The rate at which dinucleoside phosphates are cleaved by RNases is supposed to be determined by the mole fraction of enzyme-substrate complexes in which the phosphodiester moiety of a dinucleoside phosphate has a highly reactive conformation. The mole fraction of such complexes for a particular RNase depends on the nature of a nucleoside at the O5'-end of the phosphodiester bond. Experimental data are presented to support this hypothesis.

Amino acid sequence and S-S bonds of Penicillium brevicompactum guanyl-specific ribonuclease.

The primary structure of Penicillium brevicompactum guanyl-specific RNase was determined. The enzyme consists of 102 amino acid residues, Mr 10801. The 4 cysteine residues of the RNase are linked in pairs by disulfide bonds: Cys2-Cys10, Cys6-Cys101. P. brevicompactum RNase structure is similar to RNase T1; the degree of homology is 66%.

Correlative variations of the free energies for enzyme-substrate complex formation and the transition-state stabilization for RNases.

It was found for RNases of different specificities that changes in the free energy for substrate-enzyme binding induced by variations in the nucleotide base structure are accompanied by proportional changes in kcat/Km. This was considered to be a consequence of the strain in the enzyme-substrate complex.

Dimerization of deoxyribonuclease I, lysozyme and papain. Effects of ionic strength on enzymic activity.

Transition of bovine ribonuclease A from its monomeric to a dimeric form changes the pattern of enzymic activity response to ionic strength [Sorrentino, S., Carsana, A., Furia, A., Doskocil, J., and Libonati, M. (1980) Biochim. Biophys. Acta. 609, 40-52]. To see whether this phenomenon could be common to other enzyme-substrate systems, the action of various dimeric and monomeric enzymes (ox pancreas deoxyribonuclease, hog spleen acid deoxyribonuclease, bovine seminal ribonuclease, egg-white lysozyme, and papain) on polyelectrolytic substrates has been studied under different conditions of ionic strength. Dimerization of ox pancreas deoxyribonuclease, lysozyme and papain was obtained by cross-linkage with dimethyl suberimidate. The main results of the investigation, similar to those obtained with ribonuclease A, are the following. 1. Enzyme monomers and dimers show markedly different patterns of activity response to ionic strength at given pH values: the reactions catalyzed by monomeric enzymes are highly modulated by salt, whereas those catalyzed by dimeric enzymes are not. In particular, at the reaction optimum the monomeric form of an enzyme is significantly more active than the dimeric one. 2. The optimum of the reaction catalyzed by a dimeric enzyme is shifted to higher ionic strengths in comparison with that of the reaction catalyzed by a monomeric enzyme. A model is proposed that could explain these results on the basis of the influence of ionic strength on the intramolecular dynamics of the enzyme molecule and its non-specific interactions with polyelectrolytic substrates.

Guanyl-specific ribonuclease from the fungus Penicillium chrysogenum strain 152 and its complex with guanosine 3'-phosphate studied by nuclear magnetic resonance.

The method of proton magnetic resonance was used to obtain information on the active site of the guanyl-specific ribonuclease from Penicillium chrysogenum, strain 152A. Four pH-dependent signals in the aromatic region of the proton NMR spectrum of the enzyme were assigned to the C-2 and C-4 protons of the two histidine residues. To determine the pK values and the environment of the histidine residues the pH dependence of their chemical shifts was studied and experimental curves thus obtained were analyzed taking into account the effect of other dissociating groups of the enzyme. The pK values of the histidine residues were found to be equal to 7.92 +/- 0.04 and 7.86 +/- 0.09. The results of the calculations indicate that each histidine residue should interact with an acidic group (carboxylic) of the protein (pK 4.33 and 3.48) and the distance between two histidine residues does not exceed 0.85 nm. The rate constants for the quasi-first order reaction of deuterium exchange of the histidine residues (11.2 s-1 and 3.7 x-1) suggest that both residues are accessible, though to a different degree to solvent. Formation of a complex between the enzyme and guanosine 3'-phosphate (Guo3'P) is accompanied by the shift of the histidine pK toward the alkaline region by 0.5. The existence of the complex is controlled by dissociation of a histidine residue with pK 8.7 in alkaline medium and by protonation of the N-7 of Guo3'P (pK 2.4) in acid medium. Nuclear Overhauser effect measurements were used to determine the glycosidic torsion angle for the Guo3'P in the complex and to estimate the distances between the histidine residues of the enzyme and ribose ring of Guo-3'P. The results obtained suggest that the nucleotide in the complex has an anti conformation and the least exposed histidine is spaced not more than 0.5 nm from the C-1' proton of the nucleotide ribose ring. A model for the enzyme-nucleotide complex is presented.