Adenosine diphosphoribose transfer reactions catalyzed by Bungarus fasciatus venom NAD glycohydrolase

Reactions catalyzed by purified Bungarus fasciatus venom NAD glycohydrolase were demonstrated to include ADP-ribose transfer from NAD to alcohols and to imidazole derivatives to produce a variety of ADP-ribosides. The formation of products was monitored by high performance liquid chromatography. In the enzyme-catalyzed alcoholysis of NAD, the ratio of n-alkyl-ADP-riboside formed to the hydrolytic product, ADP-ribose, increased linearly with alcohol concentration. The effectiveness of alcohols as acceptors of the ADP-ribose moiety in these reactions increased with increasing chainlength of the alcohol used. Linear positive chainlength effects extended from methanol to pentanol suggesting facilitation of these reactions by nonpolar interactions. In the methanolysis reaction, NADP, thionicotinamide adenine dinucleotide, nicotinamide-1, N6-ethenoadenine dinucleotide, and 3-acetylpyridine adenine dinucleotide were shown to be as effective as NAD as donor substrates. The NAD glycohydrolase-catalyzed ADP-ribose transfer to pyridine bases to form NAD analogs was studied at pyridine base concentrations above those determined to be saturating for the base exchange reaction. Under these conditions, the ratio of base exchange to hydrolysis of NAD was directly related to the pKa of the ring nitrogen of the pyridine base employed. In addition to alcoholysis and pyridine-base exchange reactions, the snake venom enzyme was demonstrated to catalyze an ADP-ribose transfer reaction to imidazole derivatives. Arginine methyl ester was ineffective as an ADP-ribose acceptor molecule in these reactions.     

Kinetic and thermodynamic parameters for Schiff base formation of vitamin B6 derivatives with amino acids

Thermodynamic and kinetic parameters for Schiff base formation of pyridoxal 5'-phosphate and pyridoxal with epsilon-aminocaproic acid as well as of pyridoxal 5'-phosphate with L-serine were obtained in 0.1 M sodium pyrophosphate buffer as a function of temperature. Changes in enthalpy, which were determined by direct microcalorimetry, were small at 25 degrees C, but varied strongly with pH for the reaction of pyridoxal 5'-phosphate with the amino acids. In contrast to the fast Schiff base formation of pyridoxal 5'-phosphate, a very slow reaction was found for pyridoxal and epsilon-aminocaproic acid concomitant with a larger change in enthalpy. By preventing hemiacetal formation the phosphate moiety plays a crucial role.     

Influence of neighboring base sequence on mutagenesis induced by in vitro misincorporation in the lacI gene of Escherichia coli.

Genetic and electrophoretic assays of misincorporation were used to assess the effect of DNA sequence on mutagenesis arising from in vitro DNA synthesis within the lacI gene of Escherichia coli. The viral strand of a derivative of phage M13 containing the entire lacI gene was annealed with a series of synthetic oligonucleotides complementary to the N-terminal region of the lacI gene. Each primer-template was incubated with E. coli DNA polymerase I (Klenow fragment) under conditions favoring misincorporation, wherein one of the 4 dNTPs was lacking ('minus' reaction) or present at very low concentration ('micro' reaction). The extent of elongation of each primer was assessed by gel electrophoresis, and lacI mutants arising during the misincorporation reactions were detected by a transfection assay in which i- base substitutions within the in vitro synthesized strand were selectively recovered by the use of uracil-containing templates. Direct dideoxy sequencing of the '-A' reaction products and sequence analysis of i- mutant progeny revealed a vast predominance of single and non-tandem multiple base transitions. The addition of small quantities of dATP to a '-A' reaction increased the mutation yield and broadened the distribution of base substitutions along the template. We detected a general bias towards increased base substitution at template positions flanked by G.C base pairs or 5'-pyrimidine, 3'-purine nearest neighbors, although considerable site-to-site variation in the occurrence of base substitutions was seen, even within identical nearest neighbor contexts.     

DNA damage and mutations induced by arachidonic acid peroxidation

Endogenous cellular oxidation of omega6-polyunsaturated fatty acids (PUFAs) has long been recognized as a contributing factor in the development of various cancers. The accrual of DNA damage as a result of reaction with free radical and electrophilic aldehyde products of lipid peroxidation is believed to be involved; however, the genotoxic and mutation-inducing potential of specific membrane PUFAs remains poorly defined. In the present study we have examined the ability of peroxidizing arachidonic acid (AA, 20:4omega6) to induce DNA strand breaks, base modifications, and mutations. The time-dependent induction of single-strand breaks and oxidative base modifications by AA in genomic DNA was quantified using denaturing glyoxal gel electrophoresis. Mutation spectra were determined in XP-G fibroblasts and a repair-proficient line corrected for this defect by c-DNA complementation (XP-G(+)). Mutation frequencies were elevated from approximately 5- to 30-fold over the background following reaction of DNA with AA for various times. The XPG gene product was found to be involved in the suppression of mutations after extended reaction of DNA with AA. Arachidonic acid-induced base substitutions were consistent with the presence of both oxidized and aldehyde base adducts in DNA. The frequency of multiple-base substitutions induced by AA was significantly reduced upon correction for the XPG defect (14% vs 2%, P = 0.0015). Evidence is also presented which suggests that the induced frequency of multiple mutations is lesion dependent. These results are compared to published data for mutations stimulated by alpha,beta-unsaturated aldehydes identified as products of lipid peroxidation.     

Kinetics of reaction of DNA-bound Fe(III)bleomycin with ascorbate: interplay of specific and non-specific binding

The aerobic redox reaction of Fe(III)bleomycin (Blm) and ascorbate was examined in the absence of DNA and in the presence of 7.5 and 25 calf thymus DNA base pairs per-drug molecule, in order to investigate the effect of DNA binding on the properties of FeBlm activation and DNA strand cleavage. Under these successive conditions, the rate of initial reduction of Fe(III)Blm became progressively slower and biphasic. Using 7.5 base pairs per-molecule of FeBlm, 2-3 times as much drug reacted in the faster step as with the larger DNA to drug ratio. In each case, the more rapid process was identified with the reaction of high spin Fe(III)Blm-DNA. With the smaller ratio, dioxygen consumption, formation of HO(2)-Fe(III)Blm-DNA, and production of DNA strand breaks as measured by the formation of base propenal were largely rate limited by the initial reaction of ascorbate with Fe(III)Blm-DNA. After a burst of reaction with the larger ratio of base pairs to Fe(III)Blm, a small fraction of the total Fe(III)Blm, representing high spin Fe(III)Blm, entered a steady state as HO(2)-Fe(III)Blm-DNA. Thereafter, reaction of dioxygen and base propenal formation occurred slowly with similar first-order rate kinetics. In order to explain these results, it is hypothesized that the metal domain-linker of Fe(III)Blm adopts two conformations with respect to DNA. One, at specific binding sites, is relatively unreactive with ascorbate. The other, present at non-specific sites as HPO(4)-Fe(III)Blm, is readily reactive with ascorbate to generate HO(2)-Fe(III)Blm-DNA. At the larger base pair to drug ratio, movement of Fe(III)Blm between specific and non-specific sites to generate HO(2)-Fe(III)Blm is a necessary part of the mechanism of strand scission.     

Reactions of pyrzdoxal 5'-phosphate, 6-aminocaproic acid, cysteine, and penicilamine. Models for reactions of Schiff base linkages in pyridoxal 5'-phosphate-requiring enymes.

Schiff base formation, transaldimination, and reaction with aminothiols are important reactions which occur on the surface of pyridoxal-P-requiring enzymes. As a first step in assessing the role of the protein in these reactions, models for these reactions were studied in the absence of enzyme at 25 degrees, gamma/2 0.28. The reaction of 6-aminocaproic acid with pyridoxal-P to form the Schiff base N6-(P-pyridoxylidene)-aminocaproic acid was studied as a model for formation of Schiff base on the holoenzyme...     

Slow binding of retinal to rhodopsin mutants G90D and T94D

In an examination of the effect of three rhodopsin night blindness mutations on the rate of association of 11-cis-retinal with opsin, one of the mutations (G90D) was found to slow the rate of reaction by more than 80-fold. This effect does not appear to be general to night blindness mutations as the two other mutants (A292E and T94I) were not found to bind retinal with slowed kinetics. However, T94D was similar to G90D in that the rate of retinal binding was dramatically slowed. Gly90 and Thr94 are both located in the active site of the protein close to the Schiff base counterion Glu113. Thus, the slow kinetics of Schiff base formation appear to correlate with the introduction of a negative charge close to the Schiff base counterion, suggesting a possible role for Glu113 as a catalytic base in this reaction. Consistent with this model, the E113Q mutant was also found to bind retinal more slowly than the wild type.     

Development of heterogeneous base catalysts for biodiesel production

Investigations were conducted on heterogeneous base catalysts for the transesterification of oil aimed at effective production of biodiesel. Thirteen different kinds of metal oxides containing calcium, barium, magnesium, or lanthanum were prepared as catalysts. Their catalytic activities were tested for transesterification at 60 degrees C with a 6:1 molar ratio of methanol to oil and a reaction time of 10h. The calcium-containing catalysts - CaTiO3, CaMnO3, Ca2Fe2O5, CaZrO3, and CaO-CeO2 - showed high activities and approximately 90% yields of methyl ester. Furthermore, catalytic durability tests were performed by repeating the transesterification reaction several times with the calcium-containing catalysts recovered from the previous reaction mixture. It was found that CaZrO3 and CaO-CeO2 show high durability and have the potential to be used in biodiesel production processes as heterogeneous base catalysts.     

Interrelationship between Ca2+-dependent phospholipid base-exchange reaction and phospholipase D-like activity in bovine retina

Endogenous substrates (phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine) for the Ca2+-dependent base-exchange reaction were investigated using bovine retinal microsomes. The amounts of the three bases, serine, ethanolamine and choline, released from the membranes and the amount of phosphatidic acid generated in the membranes were measured in the presence of Ca2+ with or without exogenous bases. When the membranes were incubated with Ca2+ alone, the three bases were liberated into the water-soluble fractions accompanied by accumulation of phosphatidic acid, suggesting the presence of Ca2+-dependent phospholipase D-like activity. When an exogenous base was added to the reaction mixture, the liberation of the other two bases increased slightly and the formation of phosphatidic acid decreased markedly. The exogenous base also stimulated the liberation of the same base from prelabeled phospholipids. Accompanying these changes, the exogenous base was incorporated into the membrane phospholipid. With respect to pH profile, time course and metal requirements, both the base incorporation and phospholipase D-like activity were quite similar. The amount of base incorporated generally agreed with both the decreased amount of phosphatidic acid formed and the increased amount of base released. These results suggest that, beside the base-exchange reaction, phospholipase D-like activity plays an important role in Ca2+-dependent base incorporation into bovine retinal membranes.     

Role of DNA end distortion in catalysis by avian sarcoma virus integrase.

Retroviral integrase (IN) recognizes linear viral DNA ends and introduces nicks adjacent to a highly conserved CA dinucleotide usually located two base pairs from the 3'-ends of viral DNA (the "processing" reaction). In a second step, the same IN active site catalyzes the insertion of these ends into host DNA (the "joining" reaction). Both DNA sequence and DNA structure contribute to specific recognition of viral DNA ends by IN. Here we used potassium permanganate modification to show that the avian sarcoma virus IN catalytic domain is able to distort viral DNA ends in vitro. This distortion activity is consistent with both unpairing and unstacking of the three terminal base pairs, including the processing site adjacent to the conserved CA. Furthermore, the introduction of mismatch mutations that destabilize the viral DNA ends were found to stimulate the IN processing reaction as well as IN-mediated distortion. End-distortion activity was also observed with mutant or heterologous DNA substrates. However, further analyses showed that using Mn(2+) as a cofactor, processing site specificity of these substrates was also maintained. Our results support a model whereby unpairing and unstacking of the terminal base pairs is a required step in the processing reaction. Furthermore, these results are consistent with our previous observations indicating that unpairing of target DNA promotes the joining reaction.     

The mechanism of target base attack in DNA cytosine carbon 5 methylation

We measured the tritium exchange reaction on cytosine C(5) in the presence of AdoMet analogues to investigate the catalytic mechanism of the bacterial DNA cytosine methyltransferase M.HhaI. Poly(dG-dC) and poly(dI-dC) substrates were used to investigate the function of the active site loop (residues 80-99), stability of the extrahelical base, base flipping mechanism, and processivity on DNA substrates. On the basis of several experimental approaches, we show that methyl transfer is the rate-limiting pre-steady-state step. Further, we show that the active site loop opening contributes to the rate-limiting step during multiple cycles of catalysis. Target base activation and nucleophilic attack by cysteine 81 are fast and readily reversible. Thus, the reaction intermediates involving the activated target base and the extrahelical base are in equilibrium and accumulate prior to the slow methyl transfer step. The stability of the activated target base depends on the active site loop closure, which is dependent on the hydrogen bond between isoleucine 86 and the guanine 5' to the target cytosine. These interactions prevent the premature release of the extrahelical base and uncontrolled solvent access; the latter modulates the exchange reaction and, by implication, the mutagenic deamination reaction. The processive catalysis by M.HhaI is also regulated by the interaction between isoleucine 86 and the DNA substrate. Nucleophilic attack by cysteine 81 is partially rate limiting when the target base is not fully stabilized in the extrahelical position, as observed during the reaction with the Gln(237)Trp mutant or in the cytosine C(5) exchange reaction in the absence of the cofactor.     

Physical studies of chloroacetaldehyde labelled fluorescent DNA

The reaction of chloroacetaldehyde with denatured DNA produces a fluorescent DNA where both the adenine and cytosine bases are modified. The rate of modification of DNA by chloroacetaldehyde was measured using the absorption spectrum shift. The depolarization and quantum yield of native DNA and denatured DNA were investigated as a function of temperature.The melting points and the renaturation rates of a series of derivative DNA's were investigated. The melting point was decreased by 1.3°C for each base modified per 100 base pairs corresponding to a 2.8 Kcal destabilizing free energy per mismatched base pair. The renaturation rate of the derivative DNA is reduced by a factor 2 when the melting temperature is lowered by 13°C.     

Characterization of the lysyl adducts formed from prostaglandin H2 via the levuglandin pathway.

Prostaglandin H(2) has been demonstrated to rearrange to gamma-ketoaldehyde prostanoids termed levuglandins E(2) and D(2). As gamma-dicarbonyl molecules, the levuglandins react readily with amines. We sought to characterize the adducts formed by synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins with lysine. Using liquid chromatography/electrospray mass spectrometry, we found that the reaction predominantly produces lysyl-levuglandin Schiff base adducts that readily dehydrate to form lysyl-anhydrolevuglandin Schiff base adducts. These adducts were characterized by examination of their mass spectra, by analysis of the products of their reaction with sodium cyanide, sodium borohydride, and methoxylamine and by the mass spectra derived from collision-induced dissociation in tandem mass spectrometry. The Schiff base adducts also are formed on peptide-bound lysyl residues. In addition, synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins produced pyrrole-derived lactam and hydroxylactam adducts upon reaction with lysine as determined by tandem mass spectrometry. A marked time dependence in the formation of these adducts was observed: Schiff base adducts formed very rapidly and robustly, whereas the lactam and hydroxylactam adducts formed more slowly but accumulated throughout the time of the experiment. These findings provide a basis for investigating protein modification induced by oxygenation of arachidonic acid by the cyclooxygenases.     

QM/MM and free-energy simulations of deacylation reaction catalysed by sedolisin,a serine-carboxyl peptidase

Quantum mechanical/molecular mechanical free-energy simulations were performed to understand the deacylation reaction catalysed by sedolisin (a serine-carboxyl peptidase) and to elucidate the catalytic mechanism and the role of the active-site residues during the process. The results given here demonstrate that Asp170 may act as a general acid/base catalyst for the deacylation reaction. It is also shown that the electrostatic oxyanion hole interactions involving Asp170 may be less effective in transition state stabilisation for the deacylation step in the sedolisin-catalysed reaction compared to the general acid/base mechanism. The proton transfer processes during the enzyme-catalysed process were examined, and their role in the catalysis was discussed.     

Synthesis and antimicrobial activity of the Schiff base from chitosan and citral

Chitosan, a biocompatible, biodegradable, non-toxic polymer, is prepared from chitin, which is the second most naturally occurring biopolymer after cellulose. The Schiff base of chitosan was synthesized by the reaction of chitosan with citral working under high-intensity ultrasound. The effect of the molar ratio of chitosan to citral, reaction time, and temperature on the yield has been investigated. The optimal conditions were a temperature of 50 °C, a molar ratio of chitosan to citral of 1:6, and a reaction time of 10 h. The maximum yield achieved was 86.4% under optimum conditions. The structure of the Schiff base was characterized by FTIR spectroscopy, elemental analysis, and X-ray diffraction studies. The strong peaks at 1648.3 and 1610.6 cm⁻¹ are due to CN and CC stretching vibrations. The results confirmed that amino groups on chitosan reacted with citral to form the Schiff base. The antimicrobial activities of chitosan and Schiff base of chitosan were investigated against Escherichia coil, Staphylococcus aureus, and Aspergillus niger. The results indicate that the antimicrobial activity of the Schiff base increases with an increase in the concentration. It was also found that the antimicrobial activity of the Schiff base was stronger than that of chitosan.     

Twisted Schiff base intermediates and substrate locale revise transaldolase mechanism

We examined the catalytic cycle of transaldolase (TAL) from Thermoplasma acidophilum by cryocrystallography and were able to structurally characterize--for the first time, to our knowledge--different genuine TAL reaction intermediates. These include the Schiff base adducts formed between the catalytic lysine and the donor ketose substrates fructose-6-phosphate and sedoheptulose-7-phosphate as well as the Michaelis complex with acceptor aldose erythrose-4-phosphate. These structural snapshots necessitate a revision of the accepted reaction mechanism with respect to functional roles of active site residues, and they further reveal fundamental insights into the general structural features of enzymatic Schiff base intermediates and the role of conformational dynamics in enzyme catalysis, substrate binding and discrimination. A nonplanar arrangement of the substituents around the Schiff base double bond was observed, suggesting that a structurally encoded reactant-state destabilization is a driving force of catalysis. Protein dynamics and the intrinsic hydrogen-bonding pattern appear to be crucial for selective recognition and binding of ketose as first substrate.     

Binding of netropsin to DNA and synthetic polynucleotides

The interaction of netropsin with DNA and synthetic polydeoxyribonucleotides was studied by absorption spectrophotometry and circular dichroism. The results are consistent with a model in which a netropsin molecule occupies five base pairs in binding and carries three reaction sites each capable of interacting with one AT base pair. We associate these reaction sites with the antibiotic peptide groups which probably interact with AT base pairs by a hydrogen bonding mechanism.     

Reaction of DNA-bound Co(II)bleomycin with dioxygen.

The aerobic oxidation of Co(II)bleomycin bound to calf thymus DNA has been investigated in relation to the mechanism of reaction in solution in the absence of DNA. Kinetics of dioxygenation of the Co(II) complex were followed by spectrophotometric and electron spin resonance spectroscopy as well as dioxygen analysis. The reaction is slower than when carried out in solution; its rate is inversely related to the ratio of DNA base pairs to Co(II)bleomycin. The subsequent oxidation reaction, observed spectrophotometrically and by dioxygen analysis, is second order in cobalt complex. The calculated second order rate constant is also inversely related to the base pair to metal complex ratio. Once this ratio exceeds three, the reaction rate slows significantly with each additional increment of DNA added to the starting reaction mixture. Taking advantage of the high stability of O(2)-Co(II)bleomycin bound to greater than a 3-fold excess of DNA base pairs, it could be demonstrated that the rate constant for oxidation of two O(2)-Co(II)bleomycin molecules is much slower than that for O(2)-Co(II)bleomycin plus Co(II)bleomycin. With the same technique it was observed that the metal centers of O(2)-Co(II)bleomycin and Fe(II)bleomycin also undergo oxidation. The binding to DNA of both solution products of the oxidation of Co(II)bleomycin by O2 was examined by 1H NMR spectroscopy. Peroxy-Co(III)bleomycin, Form I, binds with higher affinity than Co(III)bleomycin, Form II. At lower ionic strength, the size of the DNA binding site for each form is about 2 base pairs/molecule of drug.     

The synthesis of 2-pyrimidinone nucleosides and their incorporation into oligodeoxynucleotides.

The synthesis of 1-(beta-D-2'-deoxyribosyl)-2-pyrimidinone (dK) and its 5-methyl derivative (d5) from 2'-deoxycytidine or 2'-deoxythymidine, respectively, via silver oxide oxidation of 4-hydrazinopyrimidines is described. The necessary hydrazine substituted pyrimidine nucleosides have been prepared by transamination of a protected cytidine derivative or by addition/elimination reactions to an O4-sulfonated thymidine derivative. Oxidation of the 4-hydrazino pyrimidines was complicated by a competing hydrolytic reaction which generated 2'-deoxyuridine or 2'-deoxythymidine. However, in the presence of an organic base such as triethylamine, oxidation became the predominant reaction. After suitable protection and formation of the 3'-phosphoramidite derivatives, these modified nucleosides were incorporated into seven self-complementary oligodeoxynucleotides by chemical synthesis using phosphite triester methodology. Oligodeoxynucleotides were prepared such that dA-dT and dG-dC base pairs were substituted by dA-d5 or dG-dK base pairs, respectively. Both circular dichroism spectra and thermal denaturation studies were used to characterize the modified oligodeoxynucleotides.