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.     

Acceptor activity of 4-N-acetylcytidine in the synthesis of (3'-5')-internucleotide bond catalyzed by pancreatic nuclease]

Cytidine and 4-N-acetylcytidine were compared as phosphate acceptors in dinucleoside monophosphate synthesis catalyzed by pancreatic ribonuclease with uridine-2',3'-cyclophosphate and cytidine-2',3'-cyclo phosphate as phosphate donors. Because of low solubility of 4-N-acetylcytidine in water, the synthesis was carried out in aqueus-organic media. The results obtained indicate that acetylation of the exoaminogroup of cytidine decreases its acceptor activity. For the first time uridilyl-(3'-5')-4-N-acetylcytidine and cytidilyl-(3'-5')-4-N-acetylcytidine are prepared enzymatically by pancreatic ribonuclease.     

Modification of a specific tyrosine residue of ribonuclease A with a diazonium inhibitor analog

The diazonium salt of 5'-(4-aminophenyl phosphoryl)-uridine 2'(3')-phosphate reacts stoichiometrically with pancreatic ribonuclease and modifies only one tyrosyl residue, which was identified as Tyr 73 in the amino acid sequence. The modification does not inhibit the biological activity of RNAase on ribonucleic acid, although a large change in the binding constant towards cytidine cyclic phosphate was observed. The modification can be inhibited by addition of the competitive inhibitor cytidine 2'(3')5'-diphosphate and may indicate that Tyr 73 is the most exposed residue or has a unique reactivity towards this reactive substrate analog.     

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.     

Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.     

Influence of terminal 3' phosphates or 2',3'-cyclic phosphates on the conformations of oligoriboadenylates, oligoribocytidylates, and the corresponding monomers

The circular dichroism spectra of chemically synthesized adenylate and cytidylate dinucleotides and trinucleotides bearing terminal 3' phosphates have been compared under a variety of conditions with the spectra obtained from the corresponding oligomers with 2',3'-terminal cyclic phosphate groups. Similar comparisons for the mononucleotides are also presented. Although the base stacking of an oligomer with a terminal cyclic phosphate might be expected to be greater than that of the corresponding oligomer with a 3' phosphate from charge repulsion considerations, the magnitudes of the Cotton effects in the former class are always considerably smaller than those in the latter class. This suggests a decreased stacking. The implications of these observations are discussed in light of the compelling crystallographic evidence that cytidine 2',3'-cyclic phosphate adopts an unusual sugar puckering and the syn conformation.     

The modified nucleosides of tRNAs. II. Synthesis of 2''-O-methylcytidylyl (3''-5'') cytidine.

The synthesis of 2'-O-methylcytidylyl (3'-5')cytidine by the triester method using as protecting groups, 2,2,2-trichloroethyl for phosphate hydroxyl group, p-chlorophenyoxyacetyl for 5-hydroxyl group, methoxymethylidene for 2',3'-cis-diol system, and benzoyl for the exo-amino group of cytidine is presented. The obtained product was characterised by UV, electrophoresis, chromatography and an enzymatic digestion.     

Theoretical conformation analysis of cytidine 2',3'-cyclic phosphate]

On the basis of calculations of conformational states of cytidine 2',3'-cyclic phosphate it is shown that the syn-form with a gauche-trans position of the exocyclic group C(5')H2OH is preferential.     

Inhibition of angiotensin converting enzyme by phosphoramidates and polyphosphates

N alpha-Phosphoryl-L-alanyl-L-proline is a reversible competitive inhibitor of angiotensin converting enzyme with a Ki of 1.4 nM. Alkylation of one phosphate oxygen with methyl, ethyl, or benzyl does not change the Ki. The high activity of the O-alkylated inhibitors demonstrates that the two phosphate oxygen anions do not constitute a bidentate ligand of the active site zinc ion. Substitution of valyltryptophan, glycylglycine, or delta-aminovaleric acid for alanylproline in the phosphoramidate raises the Ki to 12 nM, 25 microM, and 178 microM, respectively. Methylation of the alanine nitrogen in phosphorylalanylproline raises the Ki to 29 microM. Polyphosphates inhibit converting enzyme with the following Ki's: phosphate, approximately 300 mM; pyrophosphate, 2 mM; tripolyphosphate, 18 microM; tetrapolyphosphate, 150 microM. The inhibition by tripolyphosphate appears to be competitive and is unaffected by the addition of excess zinc ion. Since the Ki of tripolyphosphate is nearly 10-fold lower than that of N-phosphoryl-delta-aminovaleric acid and is near that of N alpha-phosphorylglycylglycine, its terminal phosphates may bind the zinc site and the cationic site on the enzyme, thus spanning the S1' and S2' sites.     

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.     

Green tea polyphenols as inhibitors of ribonuclease A

Ribonucleases (RNases), which are essential for cleavage of RNA, may be cytotoxic due to undesired cleavage of RNA in the cell. The quest for small molecule inhibitors of members of the ribonuclease superfamily has become indispensable with a growing number exhibiting unusual biological properties. Thus, inhibitors of RNases may serve as potential drug candidates. Green tea catechins (GTC), particularly its major constituent (-)-epigallocatechin-3-gallate (EGCG), have reported potential against cell proliferation and angiogenesis induced by several growth factors including angiogenin, a member of the RNase superfamily. This study reports the inhibition of bovine pancreatic ribonuclease A (RNase A) by EGCG and GTC. This has been checked qualitatively by an agarose gel based assay. Enzyme kinetic studies with cytidine 2',3' cyclic monophosphate as the substrate have also been conducted. Results indicate substantial inhibitory activity of a noncompetitive nature with an inhibition constant of approximately 80 microM for EGCG and approximately 100 microM for GTC measured in gallic acid equivalents.     

Interaction between adenosine A 2B-receptors and alpha2-adrenoceptors on the modulation of noradrenaline release in the rat vas deferens: possible involvement of a group 2 adenylyl cyclase isoform

In the prostatic portion of rat vas deferens, activation of adenosine A 2B-receptors, beta2-adrenoceptors, adenylyl cyclase or protein kinase A caused a facilitation of noradrenaline release. Blockade of alpha2-adrenoceptors with yohimbine (1 microM) attenuated the facilitation mediated by adenosine A 2B-receptors and by direct activation of adenylyl cyclase with forskolin but not that mediated by beta2-adrenoceptors or by direct activation of protein kinase A with 8-bromoadenosine-3',5'-cyclicAMP. The adenosine A 2B- and the beta2-adrenoceptor-mediated facilitation was prevented by the adenylyl cyclase inhibitors, 2',5'-dideoxy-adenosine (3 microM) and 9-cyclopentyladenine (100 microM), at concentrations that also attenuated the release enhancing effect of forskolin, but were not changed by the phospholipase C inhibitor 1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122, 1 microM). Facilitation of noradrenaline release mediated by adenosine A 2B-receptors was also attenuated by activation of protein kinase C with the phorbol ester 12-myristate 13-acetate (1 microM) and by inhibition of Gbetagamma subunits with an anti-betagamma peptide; facilitation mediated by beta2-adrenoceptors was mainly attenuated by the calmodulin inhibitor calmidazolium (10 microM) and by the calmodulin kinase II inhibitor (N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzene-sulfonamide phosphate (KN-93, 5 microM). The results suggest that adenosine A 2B- but not beta2-adrenoceptor-mediated facilitation of noradrenaline release is enhanced by an ongoing activation of alpha2-adrenoceptors. They further suggest that adenosine A 2B-receptors and beta2-adrenoceptors are coupled to distinct adenylyl cyclase isoforms what may explain the different influence of alpha2-adrenoceptor signalling pathway on the facilitatory effects mediated by the two adenylyl cyclase coupled receptors.     

Inhibition of acyl-CoA synthetase by triacsins

Triacsin A, 1-hydroxy-3-(E,E-2',4'-undecadienylidine) triazene and triacsin C, 1-hydroxy-3-(E,E,E-2',4',7'-undecatrienylidine) triazene are potent inhibitors of acyl-CoA synthetase (EC 6.2.1.3). The concentrations of triacsin A required for 50% inhibition of acyl-CoA synthetase from Pseudomonas aeruginosa and from rat liver are 17 and 18 microM, and those of triacsin C are 3.6 and 8.7 microM, respectively. Kinetic analysis indicates that inhibition of triacsin A is non-competitive with respect to the two substrates ATP and coenzyme A, but is competitive with respect to long-chain fatty acids. The apparent Ki value is 8.97 microM when oleic acid is used as substrate. Acid hydrolysis of triacsins results in corresponding polyenic aldehydes with no activity. This suggests that the N-hydroxytriazene moiety is essential for inhibitory activity against acyl-CoA synthetase.     

Analogs of (2'-5')oligo(A). Endonuclease activation and inhibition of protein synthesis in intact cells

Synthetic analogs of (2'-5')oligo(A) were assayed for endonuclease activation in cell extracts and for inhibition of protein synthesis in intact cells. The analogs are triadenylates: (i) methylated in the terminal 3'-OH; (ii) methylated at all three 3'-OH groups; (iii) with different numbers of phosphate groups at the 5' terminus or with a methylene group between the beta- and gamma-phosphate. Only 5'-phosphorylated monomethylated analogs activate an endonuclease in cell extracts and are powerful inhibitors of protein synthesis in intact cells. The analogs with only one 5'-terminal phosphate may require addition of another phosphate for activity since the kinase inhibitor 2-aminopurine prevents endonuclease activation by this compound but not by the di- and triphosphate-terminated triadenylates. These results suggest that two terminal phosphates and one or two free 3'-OH are required for endonuclease activation and inhibition of protein synthesis. The monomethylated analogs are more active than (2'-5')pppA3 because of their resistance to degradation by cellular enzymes. Accordingly, the monomethylated analogs cause a prolonged inhibition of protein synthesis in human fibroblasts treated with nanomolar concentrations of these compounds.     

Conformational studies of some 2':3'-cyclic mononucleotides in solution by different nuclear-magnetic-resonance methods

The conformations of the 2':3'-cyclic mononucleotides of adenosine and cytidine in deuterium oxide has been studied at pH 2.3, using lanthanide ions as paramagnetic nuclear magnetic resonance (NMR) probes. It was not possible to find any single conformation for these molecules which accounts for the observed shift and relaxation data. This situation is in agreement with the interpretation of vicinal 1H-1H and 1H-31P coupling constants, which indicate that the ribofuranose and cyclic phosphate rings are in rapid equilibrium between different puckered forms. The interpretation of the lanthanide data in terms of an equilibrium between different conformations give average rotamer populations in good agreement with the coupling constant analysis. The conformations of these systems in aqueous solutions were found to be more flexible than in the solid state, where rigid planar ribofuranose rings have been observed. Adenosine 2':3'-monophosphate differs from cytidine 2':3'-monophosphate at the glycosidic link.     

A new class of powerful inhibitors of monamine oxidase A

It is well established that 1-methyl-4-phenylpyridinium (MPP), the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and most of its analogs are good competitive inhibitors of monoamine oxidase A, with Ki values in the micromolar range, but they inhibit monoamine oxidase B only at much higher concentrations. We report here the finding that alkyl derivatives of MPP+ substituted at the 4' position of the aromatic ring are considerably more effective reversible inhibitors of the A type enzyme, with Ki values in the nanomolar range (0.075-1.6 microM). They inhibit the B type enzyme only at 2 to 3 orders of magnitude higher concentrations (32-374 microM).     

Molecular basis for P-site inhibition of adenylyl cyclase

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.     

Stereochemistry of internucleotidic bond formation by tRNA nucleotidyltransferase from baker's yeast.

Isomer A of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) is a substrate for tRNA nucleotidyltransferase from baker's yeast, whereas isomer B is a competitive inhibitor. The tRNA resulting from this reaction has a phosphorothioate instead of a phosphate diester linkage at the last internucleotidic linkage between cytidine and adenosine. On limited digestion of this tRNA with RNase A, one can isolate cytidine 2',3'-cyclic phosphorothioate which can be deaminated to uridine 2',3'-cyclic phosphorothioate. It can be shown that this compound is the endo isomer and that, therefore, the phosphorothioate diester bond in the tRNA must have had the R configuration. This result indicates that no racemization during the condensation of ATP alpha S, isomer A, onto the tRNA had occurred. Whether inversion or retention of configuration had taken place awaits elucidation of the absolute configuration of isomer A of ATP alpha S.     

3' Esters of ADP as energy-transfer inhibitors and probes of the catalytic site of oxidative phosphorylation.

1. A large series of 3' esters of ADP has been synthesized. Several of these can serve as photoaffinity labels; others exhibit fluorescent properties. The corresponding AMP and ATP derivatives have also been synthesized in some cases. 2. The influence of the 3'-O-acyl nucleotides on energy-linked functions of beef-heart submitochondrial particles has been investigated. The following results were obtained. a) 3'Esters of ADP are powerful and highly specific inhibitors of oxidative phosphorylation. The inhibition is competitive to ADP and Ki values as low as 0.05 microM, for the 3'-O-(1)naphthoyl ester of ADP, could be observed. b) The inhibition of oxidative phosphorylation by 3' esters of ADP appears to be non-competitive versus inorganic phosphate. c) The nucleotide analogs are not phosphorylated themselves. The corresponding ATP analogs can not drive energy-linked process. d) The 3' esters of AMP are ineffective as inhibitors, whereas the ATP derivatives are only comparatively weak inhibitors. e) Uncoupled or solubilized ATPase is almost two orders of magnitude less sensitive against inhibition by 3' esters than coupled systems. The analogs exert maximal inhibition specifically in systems involving an 'energized' state of the coupling device. f) Azido-group-bearing analogs can be used for irreversible photoinactivation of the coupling ATPase. Photoinactivation also is most efficient when carried out with 'energized' particles. g) The inhibitory properties are similar also in ATP-driven NAD+ reduction by succinate, and in the uncoupler-sensitive ATP in equilibrium with Pi exchange. The required concentrations for half-maximal inhibition are somewhat higher than in oxidative phosphorylation, but lower than with uncoupled ATPase. 3. From molecular models, from substituent properties, and from the conditions required for inhibition it is concluded that these highly effective analogs of ADP may act as conformation-specific probes at the catalytic site of oxidative phosphorylation. The results are interpreted in terms of a model suggesting that, in the process of ATP synthesis, a hydrophobic cavity on the enzyme is exposed only in the energized state, accepting the large 3' substituent. The substituent is assumed to inhibit phosphoryl transfer and/or conformational transitions inherent in the process of ADP phosphorylation by steric hinderance.     

Purification and characterization of enkephalin-degradating enzymes from calf-brain striatum

Enkephalinase A and B are extracted from Triton-X 100 washed calf-brain particles and purified by DEAE-cellulose chromatography. Both enzymes have identical Km values in their membrane-bound and soluble form. Enkephalinase A has a pH optimum at 6.9 and a Km for Leu-enkephalin of 20-25 microM, which hardly depends on the pH. Thiorphan and phosphate are purely competitive inhibitors of Enkephalinase A with Ki values of 3 nM and 1.5 mM respectively (pH = 6.85). Enkephalinase B is not affected by phosphate or thiorphan. It has a Km for Leu-enkephalin of 10 microM, a pH optimum of 7.0 and is inhibited by low concentrations of apolar dipeptides.