High-resolution crystal structures of ribonuclease A complexed with adenylic and uridylic nucleotide inhibitors. Implications for structure-based design of ribonucleolytic inhibitors

The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3',5'-ADP, 2',5'-ADP, 5'-ADP, U-2'-p and U-3'-p have been determined at high resolution. The structures reveal that each inhibitor binds differently in the RNase A active site by anchoring a phosphate group in subsite P1. The most potent inhibitor of all five, 5'-ADP (Ki = 1.2 microM), adopts a syn conformation (in contrast to 3',5'-ADP and 2',5'-ADP, which adopt an anti), and it is the beta- rather than the alpha-phosphate group that binds to P1. 3',5'-ADP binds with the 5'-phosphate group in P1 and the adenosine in the B2 pocket. Two different binding modes are observed in the two RNase A molecules of the asymmetric unit for 2',5'-ADP. This inhibitor binds with either the 3' or the 5' phosphate groups in subsite P1, and in each case, the adenosine binds in two different positions within the B2 subsite. The two uridilyl inhibitors bind similarly with the uridine moiety in the B1 subsite but the placement of a different phosphate group in P1 (2' versus 3') has significant implications on their potency against RNase A. Comparative structural analysis of the RNase A, eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and human angiogenin (Ang) complexes with these and other phosphonucleotide inhibitors provides a wealth of information for structure-based design of inhibitors specific for each RNase. These inhibitors could be developed to therapeutic agents that could control the biological activities of EDN, ECP, and ANG, which play key roles in human pathologies.     

Toward rational design of ribonuclease inhibitors: high-resolution crystal structure of a ribonuclease A complex with a potent 3',5'-pyrophosphate-linked dinucleotide inhibitor.

The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'-p [5'-phospho-2'-deoxyuridine-3'-pyrophosphate (P'-->5') adenosine 3'-phosphate] has been determined at 1.7 A resolution. This dinucleotide is the most potent low molecular weight inhibitor of RNase A reported to date (K(i) = 27 nM) and is also effective against two major nonpancreatic RNases: eosinophil-derived neurotoxin and RNase-4; in all cases, tight binding in large part derives from the unusual 3',5'-pyrophosphate internucleotide linkage [Russo, N., and Shapiro, R. (1999) J. Biol. Chem. 274, 14902-14908]. The design of pdUppA-3'-p was based on the crystal structure of RNase A complexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) [Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588]. The adenosine of pdUppA-3'-p adopts an atypical syn conformation not observed for standard adenosine nucleotides bound to RNase A. This conformation, which allows extensive interactions with Asn 67, Gln 69, Asn 71, and His 119, is associated with the placement of the 5'-beta-phosphate of the adenylate, rather than alpha-phosphate, at the site where substrate phosphodiester bond cleavage occurs. The contacts of the deoxyuridine 5'-phosphate portion of pdUppA-3'-p appear to be responsible for the 9-fold increased affinity of this compound as compared to ppA-3'-p: the uracil base binds to Thr 45 in the same manner as previous pyrimidine inhibitors, and the terminal 5'-phosphate is positioned to form medium-range Coulombic interactions with Lys 66. The full potential benefit of these added interactions is not realized because of compensatory losses of hydrogen bonds of Lys 7 and Gln 11 with the terminal 3'-phosphate and the adenylate 5'-alpha-phosphate, which were not predicted by modeling. The results reported here have important implications for the design of improved inhibitors of RNase A and for the development of therapeutic agents to control the activities of RNase homologues such as eosinophil-derived neurotoxin and angiogenin that have roles in human pathologies.     

Cleavage of 3',5'-pyrophosphate-linked dinucleotides by ribonuclease A and angiogenin

Recently, 3',5'-pyrophosphate-linked 2'-deoxyribodinucleotides were shown to be >100-fold more effective inhibitors of RNase A superfamily enzymes than were the corresponding monophosphate-linked (i.e., standard) dinucleotides. Here, we have investigated two ribo analogues of these compounds, cytidine 3'-pyrophosphate (P'-->5') adenosine (CppA) and uridine 3'-pyrophosphate (P'-->5') adenosine (UppA), as potential substrates for RNase A and angiogenin. CppA and UppA are cleaved efficiently by RNase A, yielding as products 5'-AMP and cytidine or uridine cyclic 2',3'-phosphate. The k(cat)/K(m) values are only 4-fold smaller than for the standard dinucleotides CpA and UpA, and the K(m) values (10-16 microM) are lower than those reported for any earlier small substrates (e.g., 500-700 microM for CpA and UpA). The k(cat)/K(m) value for CppA with angiogenin is also only severalfold smaller than for CpA, but the effect of lengthening the internucleotide linkage on K(m) is more modest. Ribonucleotide 3',5'-pyrophosphate linkages were proposed previously to exist in nature as chemically labile intermediates in the pathway for the generation of cyclic 2',3'-phosphate termini in various RNAs. We demonstrate that in fact they are relatively stable (t(1/2) > 15 days for uncatalyzed degradation of UppA at pH 6 and 25 degrees C) and that cleavage in vivo is most likely enzymatic. Replacements of the RNase A catalytic residues His12 and His119 by alanine reduce activity toward UppA by approximately 10(5)-and 10(3.3)-fold, respectively. Thus, both residues play important roles. His12 probably acts as a base catalyst in cleavage of UppA (as with RNA). However, the major function of His119 in RNA cleavage, protonation of the 5'-O leaving group, is not required for UppA cleavage because the pK(a) of the leaving group is much lower than that for RNA substrates. A crystal structure of the complex of RNase A with 2'-deoxyuridine 3'-pyrophosphate (P'-->5') adenosine (dUppA), determined at 1.7 A resolution, together with models of the UppA complex based on this structure suggest that His119 contributes to UppA cleavage through a hydrogen bond with a nonbridging oxygen atom in the pyrophosphate and through pi-pi stacking with the six-membered ring of adenine.     

13C NMR or coenzyme A and its constituent moieties.

Natural abundance 13C nuclear magnetic resonance spectra, in D2O, were obtained for the following: pantoyl lactone, beta-alanine, cysteamine hydrochloride, cystamine dihydrochloride, calcium pantothenate, beta-aletheine oxalate, pantetheine, pantethine, pantetheine 4'-phosphate, oxypantetheine 4'-phosphate, desulfopantetheine 4'-phosphate, N-acetyl-aminodesthiopantetheine 4'-phosphate, adenosine 2',5'-diphosphate, adenosine 3',5'-diphosphate, and coenzyme A. A complete assignment of the 13C chemical shifts in the NMR spectrum of CoA is reported. Comparison of spectra indicates that CoA most likely exists in an extended conformation.     

Dibasic amines as competitive ions improve the resolution between polyanionic nucleotides.

Aliphatic diamines when used as single ion pairing reagents were capable of resolving 3'-,5'- and 2'-,5'- nucleotidyl diphosphates from one another while conventional ion pairing reagents did not separate these positional isomers. The use of 1,2-diamines resulted in the greatest resolution while increasing spacing between the amino groups progressively reduced the resolution while increasing the retention volume. A competitive ion pairing system was also developed using triethylamine as an additional ion pairing reagent. Using this system ethylenediamine, 1,2- and 1,3-diaminopropane were nearly equivalent in their ability to resolve adenosine 3'-phosphate 5'-phosphate, from adenosine 2'-phosphate 5'-phosphate, and adenosine 3'-phosphate 5'-beta-methylenephosphosulfate (3'-mePAPS) from adenosine 2'-phosphate 5'-beta-methylenephosphosulfate (2'-mePAPS), respectively. The ability to easily resolve these positional isomers allows the use of a more simplified synthetic procedure that does not involve the use selective protecting groups to specifically phosphorylate the 2' or 3' hydroxyl group. We have used this procedure on a semipreparative scale to obtain small quantities of both mePAPS and 2'-mePAPS for use in enzymatic studies.     

Enzymatically active angiogenin/ribonuclease A hybrids formed by peptide interchange

The primary structures of the blood vessel inducing protein human angiogenin and human pancreatic ribonuclease (RNase) are 35% identical. Angiogenin catalyzes the limited cleavage of ribosomal RNA (18 and 28 S), yielding a characteristic pattern of polynucleotide products, but shows no significant activity toward conventional pancreatic RNase substrates [Shapiro, R., Riordan, J. F., & Vallee, B. L. (1986) Biochemistry 25, 3527-3532]. Angiogenin/RNase hybrid enzymes--wherein particular regions of primary structure in RNase are replaced by the corresponding segments of angiogenin--serve to explore the structural features underlying angiogenin's characteristic activities. Herein we show that synthetic angiogenin peptides, Ang(1-21) and Ang(108-123), form noncovalent complexes with inactive fragments of bovine RNase A--RNase(21-124) (i.e., S-protein) and RNase(1-118), respectively--with regeneration of activity toward conventional RNase substrates. Maximal activities for the Ang(1-21)/S-protein complex (Kd = 1.0 microM) are 52%, 45%, and 15% toward cytidine cyclic 2',3'-phosphate, cytidylyl(3'----5')adenosine, and yeast RNA, respectively. In contrast, activities of the RNase(1-118)/Ang(108-123) hybrid (Kd = 25 microM) are 1-2 orders of magnitude lower toward cyclic nucleotides and dinucleoside phosphates. However, substitution of phenylalanine for Leu-115 in Ang(108-123) increases activity up to 100-fold. Both His-13 and His-114 in the angiogenin peptides are required for activity since their substitution by alanine yields inactive complexes. Importantly, the pattern of polynucleotide products formed during cleavage of ribosomal RNA by the Ang(1-21)/S-protein hybrid shows a striking resemblance to that formed by angiogenin, demonstrating that the hybrid retains features of both angiogenin and RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth-Promoting Action of Adenosine-Containing Dinucleotide on Neuroblastoma Cells: Detection of Adenosine-Cytidine Dinucleotide (ApCp) in Neurofibroma (NF1) Extracts

Abstract: Neurofibroma type 1 tissue was investigated for the presence of growth-promoting activity on human neuroblastoma cells. The activity was isolated by gel filtration and reversed-phase column chromatographs from neurofibroma type 1 extracts. An adenosine-containing dinucleotide (adenylyl(3'-5')cytidine-3'-phosphate) was identified as one of the major components of the activities by its enzymatic fragmentation and liquid chromatography/mass spectrometry. Synthetic adenosine-containing dinucleotide derivatives such as cytidyl(3'-5')adenosine, cytidyl(2'-5')adenosine, adenylyl(3'-5')cytidine, and adenylyl(2'-5')cytidine showed a similar action. Cytidyl(3'-5')adenosine, cytidyl(2'-5')adenosine, and adenylyl(2'-5')cytidine, which are able to release a free adenosine through enzymatic hydrolysis, in particular elicited a strong activity corresponding to that of adenosine with the highest action. These results suggest that neuroblastoma cells are able to use adenosine-containing dinucleotides as well as mononucleotides for their survival and proliferation.     

The degradation of ribonucleic acid in the cotyledons of Pisum arvense

1. A ribonuclease has been partially purified from the cotyledons of germinating seed of Pisum arvense. 2. The enzyme degrades ribopolynucleotides to adenosine 3'-phosphate, guanosine 3'-phosphate and the cyclic nucleotides cytidine 2',3'-phosphate and uridine 2',3'-phosphate; no resistant ;core' remains. 3. The activity of RNA-degrading enzymes in the cotyledons increases to a maximum during the first 5 days of germination, passes through a minimum around the eighth day, and thereafter increases again. 4. Ion-exchange chromatography of methanol-soluble extracts of cotyledons revealed the presence, amongst other components, of the 2'-, 3'- and 5'-phosphates of cytidine and uridine, the 3'- and 5'-phosphates of adenosine, and guanosine 5'-phosphate. 5. Seed soaked in a solution containing [(32)P]orthophosphate gave a methanol-soluble fraction containing labelled nucleoside 5'-phosphates, but nucleoside 2'- and 3'-phosphates were not labelled. 6. It is believed that the nucleoside 2'- and 3'-phosphates arise by the action of ribonuclease on cotyledon RNA.     

Kinetic studies on degradation of nucleotides catalyzed by phosphodiesterase-phosphomonoesterase from Fusarium moniliforme

Degradation of the 2'-phosphates, 3'-phosphates, 5'-phosphates, 2':3'-cyclic phosphates, 3':5'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) of adenosine, guanosine, cytidine, and uridine catalyzed by Fusarium phosphodiesterase-phosphomonoesterase was followed by means of high performance liquid chromatography. All the nucleotides were susceptible to the enzyme to a greater or lesser degree, and the kinetic constants, Km and kcat, were determined at pH 5.3 and 37 degrees C. These constants were affected by both the nucleoside moiety and the position of the phosphate. Judged from kcat/Km, the 3'-phosphates, 2':3'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) were good substrates, whereas the 2'-phosphates, 5'-phosphates, and 3':5'-cyclic phosphates were poor substrates except for adenosine 2'-phosphate, adenosine 5'-phosphate, and cytidine 5'-phosphate, which were hydrolyzed relatively easily. Among the phosphodiesters, the 2':3'-cyclic phosphates of adenosine, guanosine, and cytidine; and the 3':5'-cyclic phosphates of adenosine and cytidine were degraded into nucleoside and inorganic phosphate without release of intermediary phosphomonoester into the medium. Other phosphodiesters were degraded stepwise releasing definite intermediates.     

Only one 3'-hydroxyl group of ppp5' A2'p5'A2'p5' A (2-5A) is required for activation of the 2-5A-dependent endonuclease

To investigate the relative importance of each of the ribose 3'-hydroxyl groups of 2-5A (ppp5' A2'p5'A2'-p5' A) in determining binding to and activation of the 2-5A-dependent endonuclease (RNase L), the 3'-hydroxyl functionality of each adenosine moiety of 2-5A trimer triphosphate was sequentially replaced by hydrogen. The analog in which the 5'-terminal adenosine was replaced by 3'-deoxyadenosine (viz. ppp5'(3'dA)-2'p5' A2'p5' A) was bound to RNase L as well as 2-5A itself and was only 3 times less potent than 2-5A as an activator of RNase L. On the other hand, when the second adenosine unit was replaced by 3'-deoxyadenosine (viz. ppp5' A2'p5'(3'dA)2'p5' A), binding to RNase L was decreased by a factor of eight relative to 2-5A trimer and, even more dramatically, there was a 500-1000-fold drop in ability to activate the 2-5A-dependent endonuclease. Finally, when the 3'-hydroxyl substituent was converted to hydrogen in the 2'-terminal residue of 2-5A, a significant increase in both binding and activation ability occurred. We conclude that only the 3'-hydroxyl group of the second (from the terminus) nucleotide residue of 2-5A is needed for effective activation of RNase L.     

Mechanism of ribonucleotide reductase from herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs

Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of [3'-3H]ADP, [3'-H]UDP, and [5-3H] UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of [3'-3H]ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of [beta-32P]ClUDP with the reductase resulted in the production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase (Harris, G., Ator, M., and Stubbe, J. A. (1984) Biochemistry 23, 5214-5225; Ator, M., and Stubbe, J. A. (1985) Biochemistry 24, 7214-7221).     

Ribonuclease "XlaI," an activity from Xenopus laevis oocytes that excises intervening sequences from yeast transfer ribonucleic acid precursors.

A ribonuclease (RNase) activity, RNase "XlaI," responsible for the excision of intervening sequences from two yeast transfer ribonucleic acid (tRNA) precursors, pre-tRNA(Tyr) and pre-tRNA(3Leu), has been purified 54-fold from nuclear extracts of Xenopus laevis oocytes. The RNase preparation is essentially free of contaminating RNase. A quantitative assay for RNase XlaI was developed, and the reaction products were characterized. RNase XlaI cleavage sites in the yeast tRNA precursors were identical to those made by yeast extracts (including 3'-phosphate and 5'-hydroxyl termini). Cleavage of pre-tRNA(3Leu) by RNase XlaI and subsequent ligation of the half-tRNA molecules do not require removal of the 5' leader or 3' trailer sequences.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of a RNA debranching activity from HeLa cells

The splicing of messenger RNA precursors (pre-mRNA) of eukaryotic cells involves the formation of a branched RNA intermediate known as a RNA lariat. This structure is formed in the first step of the reaction when a cleavage at the 5' splice site generates the 5' exon and a RNA species containing the intron and 3' exon in which the phosphate moiety at the 5' end of the intron is forming a 2'-5' phosphodiester bond with the 2'-hydroxyl moiety of a specific adenine residue near the 3' end of the intron forming a RNA branch with the following structure: -pA2'-pX-3'-pZ-. We have purified a debranching activity approximately 700-fold from the cytosolic fraction of HeLa cells. This activity catalyzes the hydrolysis of the 2'-5' phosphodiester bond of branched RNA structures yielding a 5'-phosphate end and a 2'-hydroxyl group at the branch attachment site. The activity possessed a sedimentation coefficient of 3.5 S. The reaction catalyzed by the purified fraction requires a divalent cation and is optimal at pH 7.0. The purified activity can efficiently hydrolyze triester trinucleotide structures (pY2'-pX-3'-pZ-) prepared by digestion of RNA lariats with nuclease P1. In contrast, a 2' phosphate monoester product (-pG2'-p 3'-pC-), formed by the wheat germ RNA ligase, was not attacked.     

Chemical synthesis and biological characterization of phosphorothioate analogs of 2', 5'-3'-deoxyadenylate trimer.

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate (2-5A) dependent endoribonuclease (RNase L), four 2-5A trimer analogs were examined to evaluate the effect of chirality of phosphorothioate substitution on biological activity. The chemical syntheses and purification of the four isomers of P-thio-3'-deoxyadenylyl-(2'-5')-P-thio-3'- deoxyadenylyl-(2'-5')-3'-deoxyadenosine, by the phosphoramidite approach, is described. The isolated intermediates were characterized by elemental and spectral analyses. The fully deblocked compounds were characterized by 1H and 31P NMR and HPLC analyses. The 2',5'-(3'dA)3 cores with either Rp or Sp chirality in the 2',5'-internucleotide linkages will bind to but will not activate RNase L. This is in contrast to 2',5'-A3 core analogs with either RpRp or SpRp phosphorothioate substitution in the 2',5'-internucleotide linkages which can bind to and activate RNase L. There are also marked differences in the ability of the 2',5'-A3 analogs to activate RNase L following introduction of the 5'-monophosphate. For example, the 5'monophosphates of 2',5'-(3'dA)3-RpRp and 2',5'-(3'dA)3-SpRp can bind to and activate RNase L, whereas the 5'-monophosphates of 2',5'-(3'dA)3-RpSp and 2',5'-(3'dA)3-SpSp can bind to but can not activate RNase L.     

Fluorescence studies of ATP-diphosphohydrolase from Solanum tuberosum var. Desirée

Chemical modification of potato apyrase suggests that tryptophan residues are close to the nucleotide binding site. Kd values (+/- Ca2+) for the complexes of apyrase with the non-hydrolysable phosphonate adenine nucleotide analogues, adenosine 5'-(beta,gamma-methylene) triphosphate and adenosine 5'-(alpha,beta-methylene) diphosphate, were obtained from quenching of the intrinsic enzyme fluorescence. Other fluorescent nucleotide analogues (2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-diphosphate. 1,N6-ethenoadenosine triphosphate and 1,N6-ethenoadenosine diphosphate) were hydrolysed by apyrase in the presence of Ca2+, indicating binding to the active site. The dissociation constants for the binding of these analogues were calculated from both the decrease of the protein (tryptophan) fluorescence and enhancement of the nucleotide fluorescence. Using the sensitised acceptor (nucleotide analogue) fluorescence method, energy transfer was observed between enzyme tryptophans and ethene-derivatives. These results support the view that tryptophan residues are present in the nucleotide-binding region of the protein, appropriately oriented to allow the energy transfer process to occur.     

Synthesis and template-directed polymerization of adenylyl(3'-5')adenosine cyclic 2', 3'-phosphate.

Adenylyl(3'-5')adenosine cyclic 2',3'-phosphate (A-A greater than p) was synthesized and its polymerization was attempted under various conditions inthe presence of poly(uridylic acid) and1,3-propanediamine. Reaction at -20 degrees C for 16 days gave polymerized products (up to the 8-mer) in 15% yield and was proved to be dependent on the template. Reaction at 0 degrees C for 16 days gave more extensive (up to the 10-mer) and more efficient (35%) polymerization. The newly formed phosphodiester linkage was exclusively 2'-5'. These results are discussed in comparison with the monomer-condensation reaction.     

Potent inhibition of mammalian ribonucleases by 3', 5'-pyrophosphate-linked nucleotides.

Molecular modeling based on the crystal structure of the complex of bovine pancreatic RNase A with the inhibitor 5'-diphosphoadenosine 3'-phosphate (ppAp) (Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588) was used to design new inhibitors that extend into unoccupied regions of the enzyme active site. These compounds are dinucleotides that contain an unusual 3',5'-pyrophosphate linkage and were synthesized in solution by a combined chemical and enzymatic procedure. The most potent of them, 5'-phospho-2'-deoxyuridine 3'-pyrophosphate, P' --> 5'-ester with adenosine 3'-phosphate (pdUppAp), binds to RNase A with Ki values of 27 and 220 nM at pH 5.9 and 7, respectively. These values are 6-9-fold lower than those for ppAp and 50-fold lower than that for the transition state analogue, uridine vanadate. pdUppAp has broad specificity; it is an effective inhibitor of at least two other members of the pancreatic RNase superfamily, human RNase-2 (eosinophil-derived neurotoxin) and RNase-4, which share only 36-44% sequence identity with the pancreatic enzyme. The potency of pdUppAp and the other inhibitors described here depends critically on the extended internucleotide linkage; the pyrophosphate group enhances dinucleotide binding to the three RNases by 2.1-2.9 orders of magnitude, as compared with a monophosphate. These data give further insight into the organization of the catalytic centers of the various RNases. Moreover, the new class of inhibitors provides a useful means by which to probe the biological actions of these and other related enzymes.     

The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives

The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.     

Circularization of linear viroid RNA via 2'-phosphomonoester, 3', 5'-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas.

A novel type of RNA ligase activity in extracts of wheat germ or Chlamydomonas requires 2', 3'-cyclic phosphate and 5'-phosphate ends for ligation to form a 2'-phosphomonoester, 3',5'-phosphodiester bond. Using 5'-3 2P-labeled linear PSTV, we demonstrate that RNase T1-nicked viroid predominantly forms (formula; see text) U-bonds. Natural linear PSTV, however, forms mainly (formula; see text) A-bonds upon enzymatic circularization. We show that natural linear PSTV RNA has nicks between C181 and A182, or between C348 and A349, and that consequently C181 and C348 carry 2',3'-cyclophosphate termini.