Reactions of lumiflavin and lumiflavin radicals with .CO2- and alcohol radicals

The kinetics of reaction of oxidized lumiflavin (F0) with the radicals .CO2(-), CH3CHOH, and (CH3)2COH have been investigated at pH 7 and 24 +/- 1 degree C by the pulse radiolysis technique. The radicals have been shown to react with lumiflavin with second-order rate constants of 36 +/- 4, 26 +/- 3, and 20 +/- 3 in units of 10(8) M-1 s-1, respectively. These rate constants are close to the diffusion limit. The main product in each case was the lumiflavin semiquinone radical FH.. By utilization of long pulses (approximately 100 mus), it was shown that the reaction FH. + .AH(alpha) leads to FH- + A(alpha) + H+ [.AH(alpha) = .CO2(-), CH3CHOH, or (CH3)2COH] proceeded for all three types of .AH(alpha) radical with second-order rate constants of 17 (+4,-3), 9 (+5,-3), and 9 (+4,-3), respectively, in the above units. The beta-carbon radical .CH2CH(OH)CH3 added to .FH, forming an alkylated flavin, while the .CH2CH2OH radical appeared to be capable of addition or hydrogen atom donation to .FH.     

Effect of phosphate ions on the fluorescence of tryptophan derivatives. Implications in fluorescence investigation of protein-nucleic acid complexes

In order to test the ability of phosphate groups to quench the fluorescence of tryptophan in protein-nucleic acid complexes we have studied the effect of various phosphate ions on the fluorescence of tryptophan derivatives. Unsubstituted and monoalkyl monoanions (H2PO4- and CH3OPO3H-) quench the fluorescence of all investigated indole derivatives while the dimethyl anion (CH3O)2 PO2- does not. This suggests that quenching of tryptophan fluorescence by phosphate monoanions requires the presence of an acidic OH group and could be due to a proton transfer from the phosphate ion to the indole chromophore. Trianions (PO4 3-4) which are strong proton acceptors quench the fluorescence of all tryptophan derivatives except N(1)methyl tryptophan. This result strongly supports our proposal that quenching of tryptophan fluorescence by phosphate trianions occurs through deprotonation of the NH indole group. Bianions (HPO '4(7), and CH3O PO3 2-3) quench the fluorescence of several indole derivatives including N-acetyl tryptophanamide but have no effect on tryptophan or N(1)-methyl tryptophan. From our results we conclude that phosphate groups of nucleic acids are not able to quench the fluorescence of tryptophyl residues in protein-nucleic acid complexes except if an accessible residue is located near a phosphorylated polynucleotide chain end.     

OH-induced free radicals in 3'-UMP and poly(U): spin-trapping and radical chromatography

Characterization of OH-induced free radicals using 3'-UMP and poly(U) was performed by a method combining spin-trapping and radical chromatography. A N2O-saturated aqueous solution containing 3'-UMP and 2-methyl-2-nitrosopropane as a spin-trap was X-irradiated. The spin adducts generated by the reactions of OH radicals with 3'-UMP were separated by paired-ion HPLC and the separated spin adducts were identified by ESR spectroscopy. In the case of poly(U), the spin adducts were digested to oligonucleotides with RNase A and then separated and identified in the same manner as 3'-UMP. The free radicals observed for poly(U) were identical to those for 3'-UMP. The 5-yl radical and the 6-yl radical were identified as precursors of various oxidized products of the base moiety, and the 4'-yl radical and 5'-yl radical, formed by H-abstraction at the C-4' and C-5' positions of the sugar moieties, respectively, were identified as precursors of strand breaks. The 1'-yl radical, produced by H-abstraction at the C-1' position of the sugar moiety, was also identified. From the similarity of the free radicals of 3'-UMP and poly(U), it is suggested that the reactivities of OH radicals with nucleotides are identical to those in polynucleotides.     

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.     

L-methionine decarboxylase from Dryopteris filix-mas: purification, characterization, substrate specificity, abortive transamination of the coenzyme, and stereochemical courses of substrate decarboxylation and coenzyme transamination

L-Methionine decarboxylase from the male fern Dryopteris filix-mas has been purified 256-fold from acetone powder extracts to very near homogeneity. The enzyme is membrane-associated and requires detergent for solubilization during the initial extraction. The enzyme is a homodimer of subunit Mr 57,000 and shows a pH optimum at approximately 5.0 with 20 mM (2S)-methionine as substrate. The specific activity, kcat, for methionine is approximately 50 mol s(-1) (mol of active site)(-1) at pH 4.5 and below. A wide range of straight- and branched-chain (2S)-alkylamino acids are substrates for the enzyme. The values for the rate of decarboxylation, Vmax, and for the apparent Michaelis constant, Km, however, vary with structure and with the chirality at C-3. The pH dependence of V and V/K has been examined for three substrates: (2S)-methionine, valine, and leucine. Pyridoxal 5'-phosphate (PLP) is required for activity, and in the absence of excess PLP, the activity of the enzyme in incubations reduced with respect to time. The addition of PLP fully restores the activity, indicating that an abortive decarboxylation-transamination accompanies the normal decarboxylation reaction. The occurrence of the abortive reaction was confirmed by showing that [35S]methionine is converted to labeled 3-(methylthio)propionaldehyde while [4'-3H]PLP is converted to labeled pyridoxamine 5'-phosphate (PMP). The decarboxylation of (2S)-methionine gave 3-(methylthio)-1-aminopropane. Preparation of the N-camphanamide derivative of the amine allowed the C-1 methylene protons to be distinguished by 1H NMR spectroscopy. Synthetic samples of the camphanamide were prepared in which each of the C-1 methylene protons was replaced by deuterium. When (2S)-methionine and the C-2 deuteriated isotopomer were incubated with the enzyme in deuterium oxide and protium oxide, respectively, and the products were converted to their camphanamide derivatives and analyzed by 1H NMR spectroscopy, it was evident that decarboxylation occurred with retention of configuration at C-2. When the decarboxylation of six other substrates was studied, examination of the N-camphanamide derivatives of the amines indicated that decarboxylation occurred stereospecifically and, by analogy, with retention of configuration at C-2. When tritiated pyridoxal phosphate was incubated with the enzyme, tritiated pyridoxamine phosphate was formed. Analysis of the chirality of the methylene group at C-4' indicated that, during abortive transamination, protonation occurred from the 4'-si face of the coenzyme, the same stereochemical result as that obtained for several bona fide transaminase enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oligonucleotide studies. Optical rotatory dispersion of five homodinucleotides

1. The optical rotatory dispersion (ORD) of five homodinucleotides, ApAp(3'), CpCp(3'), GpGp(3'), IpIp(3') and UpUp(3') (where A, C, G, I and U represent adenosine, cytidine, guanosine, inosine and uridine respectively, and p to the left of a nucleoside symbol indicates a 5'-phosphate and to the right it indicates a 3'-phosphate), were measured as a function of pH, ionic strength and Mg(2+) concentration. 2. The ORD titrations of ApAp(3') and CpCp(3'), which were made by measuring the ORD curves at closely spaced pH intervals, exhibit a maximum at approx. pH5.0 and 5.7 for ApAp(3') and CpCp(3') respectively in the profile of the magnitude of the first Cotton effect versus pH. The results indicate that the conformational rigidity of these dinucleotides depends on the ionization state of a 3'-terminal phosphate group. 3. ApAp(3') was shown to exist as an approximately 1:1 equilibrium mixture of the two major ionic species represented by Ap((-1))Ap((-1)) and Ap((-1))Ap((-2)) at pH6.16, whereas at pH7.5 it exists exclusively as a form of Ap((-1))Ap((-2)). 4. To ascertain the effects of the presence of a terminal phosphate group and of the ionization of the secondary phosphate on the conformation of adenylate dimer, we measured the ORD of ApA, ApAp(3')CH(3) and ApAp(2'). The rotatory power of the first Cotton effect in the above series of dinucleotides decreased at 20 degrees in the order ApA> ApAp(3')CH(3) approximately ApAp(3')((-1))> ApAp(2') at pH7> ApAp(3') at pH7. 5. The pH-rotation profiles were also obtained for ApAp(2'), CpCp(2') and UpUp(3'), but no corresponding maximum was observed. Although simple nearest-neighbour calculations based on the ORD data of IpIp(3') and 5'-IMP account for the observed ORD spectrum of polyinosinic acid at low salt concentration, there were large discrepancies between calculated and experimental results of the polyguanylic acid ORD even at low ionic strength. 6. The extent to which the amplitude of the Cotton effects of IpIp(3') increases with salt concentration, especially by the addition of Mg(2+), was much greater than that observed for ApAp(3'). The implication of such salt effects on the ORD is considered.     

Chemical modification by pyridoxal 5''-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite of bovine pancreatic ribonuclease A.

Steric and chemical evidence had previously shown that residues Lys-7 and/or Arg-10 of bovine pancreatic RNAase A could belong to the p2 phosphate-binding subsite, adjacent to the 3' side of the main site p1. In the present work chemical modification of the enzyme with pyridoxal 5'-phosphate and cyclohexane-1,2-dione was carried out in order to identify these residues positively as part of the p2 site. The reaction with pyridoxal 5'-phosphate yields three monosubstituted derivatives, at Lys-1, Lys-7 and Lys-41. A strong decrease in the yield of derivatives at Lys-7 and Lys-41 was observed when either p1 or p2 was specifically blocked by 5'-AMP or 3'-AMP respectively. These experiments indicate that both sites are needed for the reaction of pyridoxal 5'-phosphate with RNAase A to take place. The positive charge in one of the sites interacts with the phosphate group of pyridoxal 5'-phosphate, giving the proper orientation to the carbonyl group, which then reacts with the lysine residue present in the other site. The absence of reaction between pyridoxal 5'-phosphate and an RNAase derivative that has the p2 site blocked supports this hypothesis. Labelling of Lys-7 with pyridoxal 5'-phosphate has a more pronounced effect on the kinetics with RNA than with the smaller substrate 2',3'-cyclic CMP. In addition, when the phosphate moiety of the 5'-phosphopyridoxyl group was removed with alkaline phosphatase the kinetic constants with 2',3'-cyclic CMP returned to values very similar to those of the native enzyme, whereas a higher Km and lower Vmax. were still observed for RNA. This indicates that this new derivative has recovered a free p1 site and, hence, the capability to act on 2',3'-cyclic CMP, but the presence of the pyridoxyl group bound to Lys-7 is still blocking a secondary phosphate-binding site, namely p2. Finally, reaction of cyclohexane-1,2-dione at Arg-10 is suppressed in the presence of 3'-AMP but only a 19% decrease is observed with 5'-AMP, suggesting that Arg-10 is also close to the p2 phosphate-binding subsite.     

Detection of 2-deoxy-D-ribose radicals generated by the reaction with the hydroxyl radical using a rapid flow-ESR method

ESR spectrum of the short-lived radicals derived from 2-deoxy-D-ribose by the reaction with the hydroxyl radical (HO*) was measured using a rapid flow method. A dielectric mixing resonator was used for the measurement, which made it possible to measure the highly sensitive ESR spectra of the radicals with a lifetime of the order of milliseconds. A complex spectrum was obtained and the spectral simulation was done to show that it was the superposition of the signals due to five radicals (I-V). Three of them were those formed by the dehydrogenation with the HO* at C-1 (I), C-3 (II), and C-4 (III) positions of the 2-deoxy-D-ribose molecule. The other two (IV and V) were carbonyl-conjugated radicals formed by the elimination of a water molecule from III and II. The results showed that dehydrogenation occurred randomly at the positions where hydroxyl groups are attached, but the most preferred position was C-3 and the radical position moved from C-3 to C-4 by the elimination of water molecule.     

Reactions of the hydrated electron with pyrimidine nucleosides halogenated at the sugar moiety: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane

Free radicals produced by the reactions of hydrated electrons with pyrimidine nucleosides halogenated at the sugar moiety (2'-chloro-2'-deoxyuridine and 2'-chlorothymidine) were studied by e.s.r. and spin-trapping. 2-Methyl-2-nitrosopropane was used as the spin-trap. The usual spin-trapping technique was extended to frozen and deoxygenated systems to avoid contamination of the trapped radicals with side-products by spin-trapping 2-methyl-2-nitrosopropane itself. When this method was applied to 2'-chloro-2'-deoxyuridine, a free radical at the C-2' position of the sugar moiety was spin-trapped together with a free radical at the C-5 position of the base moiety. This indicates that hydrated electrons both add to the base moiety and eliminate halogen anions from the halogenated sugar moiety. In the case of 2'-chlorothymidine, however, only a free radical attributed to H-addition at the C-6 position of the thymine base was observed. No radicals produced by the reaction of hydrated electrons with the halogenated sugar could be spin-trapped.     

Cyclases of the 3'-terminal phosphate in RNA: a new family of RNA processing enzymes conserved in eucarya, bacteria and archaea.

The 2',3'-cyclic phosphate termini are produced, as either intermediates or final products, during RNA cleavage by many different endoribonucleases. Likewise, ribozymes such as hammerheads, hairpins, or the hepatitis delta ribozyme, generate 2',3'-cyclic phosphate ends. Discovery of the RNA 3'-terminal phosphate cyclase has indicated that cyclic phosphate termini in RNA can also be produced by an entirely different mechanism. The RNA 3'-phosphate cyclase converts the 3'-terminal phosphate in RNA into the 2',3'-cyclic phosphodiester in the ATP-dependent reaction which involves formation of the covalent cyclase-AMP and the RNA-N3' pp5' A intermediates. The findings that several eukaryotic and prokaryotic RNA ligases require the 2',3'-cyclic phosphate for the ligation of RNA molecules raised a possibility that the RNA 3'-phosphate cyclase may have an anabolic function in RNA metabolism by generating terminal cyclic groups required for ligation. Recent cloning of a cDNA encoding the human cyclase indicated that genes encoding cyclase-like proteins are conserved among Eucarya, Bacteria, and Archaea. The protein encoded by the Escherichia coli gene was overexpressed and shown to have the RNA 3'-phosphate cyclase activity. This article reviews properties of the human and bacterial cyclases, their mechanism of action and substrate specificity. Possible biological functions of the enzymes are also discussed.     

Vibrational analysis of nucleic acids. V. Force field and conformation-dependent modes of the phosphodiester backbone modeled by diethyl phosphate.

A generalized valence force field is derived for the diethyl phosphate anion [(CH3CH2O)2PO2-] and its deuterium [(CH3CD2O)2PO2-, (CD3CH2O)2PO2- and (CD3CD2O)2PO2-] and carbon-13 [(CH3 13CH2O)2PO2-] derivatives in the stable trans-gauche-gauche-trans conformation. Normal coordinate analysis of the trans-gauche-gauche-trans conformer, which serves as a structural analog of the nucleic acid phosphodiester group, is based on comprehensive infrared and Raman spectroscopic data and vibrational assignments obtained for the diethyl phosphate anion. The generalized valence force field is in good agreement with the scaled ab initio force field of diethyl phosphate and represents significant improvement over earlier modeling of the phosphodiester moiety with dimethyl phosphate. The conformational dependence of skeletal C-C-O-P(O2-)-O-C-C stretching vibrations is also explored. Starting with the trans-gauche-gauche-trans conformation, the frequency dependence of skeletal stretching modes has been obtained by stepwise rotation of the torsion angles of the P-O and C-O bonds corresponding to nucleic acid torsions alpha (P-O5'), beta (O5'-C5'), epsilon (C3'-O3'), and zeta (O3'-P). Both symmetric and antisymmetric phosphoester stretching modes are highly sensitive to P-O and C-O torsions, whereas symmetric and antisymmetric phosphodioxy (PO2-) stretching modes are less sensitive. The present results provide an improved structural basis for understanding previously developed empirical correlations between vibrational marker bands and nucleic acid backbone conformation.     

A new affinity reagent for the site-specific, covalent attachment of DNA to active-site nucleophiles: application to the EcoRI and RsrI restriction and modification enzymes.

A modified oligodeoxyribonucleotide duplex containing an unnatural internucleotide trisubstituted 3' to 5' pyrophosphate bond in one strand [5'(oligo1)3'-P(OCH3)P-5'(oligo2) 3'] reacts with nucleophiles in aqueous media by acting as a phosphorylating affinity reagent. When interacted with a protein, a portion of the oligonucleotide [--P-5'(oligo2)3'] becomes attached to an amino acid nucleophilic group through a phosphate of the O-methyl-modified pyrophosphate linkage. We demonstrate the affinity labeling of nucleophilic groups at the active sites of the EcoRI and RsrI restriction and modification enzymes with an oligodeoxyribonucleotide duplex containing a modified scissile bond in the EcoRI recognition site. With the EcoRI and RsrI endonucleases in molar excess approximately 1% of the oligonucleotide becomes attached to the protein, and with the companion methyltransferases the yield approaches 40% for the EcoRI enzyme and 30% for the RsrI methyltransferase. Crosslinking proceeds only upon formation of a sequence-specific enzyme-DNA complex, and generates a covalent bond between the 3'-phosphate of the modified pyrophosphate in the substrate and a nucleophilic group at the active site of the enzyme. The reaction results in the elimination of an oligodeoxyribonucleotide remnant that contains the 3'-O-methylphosphate [5'(oligo1)3'-P(OCH3)] derived from the modified phosphate of the pyrophosphate linkage. Hydrolysis properties of the covalent protein-DNA adducts indicate that phosphoamide (P-N) bonds are formed with the EcoRI endonuclease and methyltransferase.     

Enzymic reactions of phosphate analogs.

A variety of phosphonates (XPO32-; X = H-, CH3-, CL3C-, CH3CH2-, and phenyl-) as well as methylarsonate have been shown to be suitable phosphate analogs for the reactions catalyzed by yeast glyceraldehyde-3-phosphate dehydrogenase and calf spleen purine nucleoside phosphorylase. The reactivity of the phosphate analogs with these two enzymes is independent of the pKa of the analog.     

Study of the structure of the duplex (Phn-NH(CH2)2NH)pd(CCAAACA) .pd(TGTTTGGC) with covalently bound 10-(2-hydroxyethyl)phenazine in an aqueous solution by 2D-1H-NMR spectroscopy]

The spatial structure of duplex (Phn-NH(CH2)2NH)pd(CCAAACA).pd(TGTTTGGC) having a N-(2-oxyethyl)-phenazinium residue covalently linked with the 5'-terminal phosphate of the heptanucleotide was studied by means of one- and two-dimensional 1H-NMR spectroscopy. The resonances of phenazinium protons, ethylenediamine linker protons, as well as, oligonucleotide H5/H6/H8/CH3 base protons and H1',H2'a, H2'b, H3', H4' deoxyribose protons have been assigned by means of 1H-COSY, 1H-NOESY and 1H-13C-COSY. The presence of the phenazine residue in duplex causes an additional imino proton signal of the terminal (G-7).(C-1) base pair, suggesting a higher stability of the duplex (Phn-NH(CH2)2NH)pd(CCAAACA).pd(TGTTTGGC) as compared to the unmodified duplex pd(CCAAACA).pd(TGTTTGGC). Analysis of NOE interactions between protons of the dye and the oligonucleotides show the phenazinium polycyclic system to intercalate between G-7 and C-8 residues of the octanucleotide.     

The reaction of solvated electrons with cytosine, 5-methyl cytosine and 2'-deoxycytidine in squeous solution. The reaction of the electron adduct intermediates with water, p-nitroacetophenone and oxygen. A pulse spectroscopic and pulse conductometric study.

Using conductivity detection, pulse radiolysis experiments showed that solvent protonation of the electron adducts of cytosine, 5-methyl cytosine and 2'-deoxycytidine occurs with rate constants k greater than or equal to 2 x 10(4) M-1S-1. The protonated electron adducts transfer an electron to p-nitroactetophenone (PNAP) with rate constants ranging from 3.5 x 10(9) to 5.3 x 10(9) M-1S-1. The transfer is quantitative (G = 2.7), as shown by conductometric and spectroscopic measurements. In the presence of O2 no electron transfer to O2 takes place, implying that O2 adds to the protonated electron adduct radicals. No electron transfer from the H- and OH-adducts of the cytosine derivatives, either to PNAP or to O2, takes place near neutral pH. It is suggested that the differences in the reaction behaviour of the H-adduct radicals and the protonated electron adduct radicals towards PNAP can be accounted for if different radicals are formed by H-addition and protonation of the electron adduct. The H atoms most probably add to the C-5-C-6 double bonds, whereas the electron adducts are protonated at N-3 and/or 0-2.     

Chromatin 3''-phosphatase/5''-OH kinase cannot transfer phosphate from 3'' to 5'' across a strand nick in DNA.

Rat liver chromatin contains a 3'-phosphatase/5'-OH kinase which may be involved in the repair of DNA strand breaks limited by 3'-phosphate/5'-OH ends. In order to determine whether the phosphate group can be transferred directly from the 3' to the 5' position, a polynucleotide duplex was synthesized between poly (dA) and oligo (dT) segments which had 3'-[32P]phosphate and 5'-OH ends. The oligo (dT) segments were separated by simple nicks as shown by the ability of T4 DNA ligase to seal the nick after the 3'-phosphate was removed by a phosphatase and the 5' end was phosphorylated with a kinase. The chromatin 3'-phosphatase/5'-OH kinase was unable to transfer phosphate directly from the 3' to the 5' end of the oligo (dT) segments in the original duplex; ATP was needed to phosphorylate the 5'-OH end. It is concluded that the chromatin 3'-phosphatase/5'-OH kinase is unable to convert a 3'-phosphate/5'-OH nick which cannot be repaired by DNA ligase directly into a 3'-OH/5'-phosphate nick which can be repaired by DNA ligase; the chromatin enzyme rather acts in two steps: hydrolysis of the 3'-phosphate followed by ATP-mediated phosphorylation of the 5'-OH end.     

Inhibition of endoribonuclease VI from Artemia larvae by cytidine 2'-phosphate

The endoribonuclease VI from Artemia larvae is non-competitively inhibited by cytidine 2'-phosphate with a Ki ca 1 microM. Neither of the cytidine monophosphates isomers with the phosphate group in the 3' or 5' position nor the cyclic 2':3' phosphate are inhibitors at concentrations up to 100 microM. Adenosine, guanosine and uridine 2' or 3' phosphates are also ineffective in this range of concentrations. Certain polyribonucleotides are potent competitive inhibitors of the ribonuclease activity.     

Exchange mechanisms for hydrogen bonding protons of cytidylic and guanylic acids.

The pH dependence of buffer catalysis of exchange of the C-4 amino protons of cyclic cytosine 2',3'-monophosphate (cCMP) and the N-1 proton of cyclic guanosine 2',3'-monophosphate (cGMP) conforms to an exchange mechanism, in which protonation of the nucleobases at C(N-3) AND G(N-7) establishes the important intermediates at neutral to acidic pH. Rate constants for transfer of the G(N-1) proton to H2O, OH-, phosphate, acetate, chloracetate, lactate, and cytosine (N-3) were obtained from 1H nuclear magnetic resonance line width measurements at 360 MHz and were used to estimate the pK or acidity of the exchange site in both the protonated and unprotonated nucleobase. These estimates reveal an increase in acidity of the G(N-1) site corresponding to 2 to 3 pK units as the G(N-7) site is protonated: At neutral pH the G(N-1) site of the protonated purine would be ionized (pK = 6.3). Determinations of phosphate, imidazole, and methylimidazole rate constants for transfer of the amino protons of cCMP provide a more approximate estimate of pK = 7 to 9 for the amino of the protonated pyrimidine. A comparison of the intrinsic amino acidity in the neutral and protonated cytosine is vitiated by the observation that OH- catalyzed exchange in the neutral base is not diffusion limited. This leads to the conclusion that protonation of the nucleobase effects a qualitative increase in the ability of the amino protons to form hydrogen bonds: from very poor in the neutral base to "normal" in the conjugate acid.     

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.     

C-1' hydrogen abstraction of deoxyribose in DNA strand scission by dynemicin A.

Dynemicin A, which is a hybrid antitumor antibiotic containing anthraquinone and enediyne cores, abstracts the C-1' hydrogen of DNA deoxyribose and then the damaged DNA leads to strand breaks with the formation of 5'- and 3'-phosphate termini. The lesions of C-4' hydrogen also occur at 3' side of G.C base pairs (i. e., 5'-CT and 5'-GA), leading to 5'-phosphate and 3'-phosphoglycolate termini or 4'-hydroxylated abasic sites. The C-1' hydrogen abstraction by dynemicin A is distinct from the preferential C-5' hydrogen abstraction of calicheamicin and neocarzinostatin.