Novel Synthesis and Anti-HIV Activity of 4′-Branched Exomethylene Carbocyclic Nucleosides Using a Ring-Closing Metathesis of Triene

The exomethylene of 6 was successfully constructed from the aldehyde 5 using Eschenmoser's reagents. A triene compound 7 was cyclized successfully using Grubbs’ II catalyst to give an exomethylene carbocycle nucleus for the target compound. A Mitsunobu reaction was successfully used to condense the natural bases (adenine, thymine, uracil, and cytosine). The synthesized cytosine analogue 20 showed moderate anti-HIV activity (EC₅₀ = 10.67 μM).     

Mechanism of nitrite-stimulated catalysis by lactoperoxidase.

The reactions of lactoperoxidase (LPO) intermediates compound I, compound II and compound III, with nitrite (NO2(-)) were investigated. Reduction of compound I by NO2(-) was rapid (k2 = 2.3 x 10(7) M(-1) x s(-1); pH = 7.2) and compound II was not an intermediate, indicating that NO2* radicals are not produced when NO2(-) reacts with compound I. The second-order rate constant for the reaction of compound II with NO2(-) at pH = 7.2 was 3.5 x 10(5) M(-1) x s(-1). The reaction of compound III with NO2(-) exhibited saturation behaviour when the observed pseudo first-order rate constants were plotted against NO2(-) concentrations and could be quantitatively explained by the formation of a 1 : 1 ratio compound III/NO2(-) complex. The Km of compound III for NO2(-) was 1.7 x 10(-4) M and the first-order decay constant of the compound III/ NO2(-) complex was 12.5 +/- 0.6 s(-1). The second-order rate constant for the reaction of the complex with NO2(-) was 3.3 x 10(3) M(-1) x s(-1). Rate enhancement by NO2(-) does not require NO2* as a redox intermediate. NO2(-) accelerates the overall rate of catalysis by reducing compound II to the ferric state. With increasing levels of H2O2, there is an increased tendency for the catalytically dead-end intermediate compound III to form. Under these conditions, the 'rescue' reaction of NO2(-) with compound III to form compound II will maintain the peroxidatic cycle of the enzyme.     

Presence of Escherichia coli of a deaminase and a reductase involved in biosynthesis of riboflavin.

Two enzymes have been partially purified from extracts of Escherchia coli B which together catalyze the conversion of the product of the action of GTP cyclohydrolase II, 2,5-diamino-6-oxy-4-(5'-phosphoribosylamine)pyrimidine, to 5-amino-2,6-dioxy-4-(5'-phosphoribitylamine)pyrimidine. These two compounds are currently thought to be intermediates in the biosynthesis of riboflavin. The enzymatic conversion occurs in two steps. The product of the action of GTP cyclohydrolase II first undergoes hydrolytic deamination at carbon 2 of the ring, followed by reduction of the ribosylamino group to a ribitylamino group. The enzyme which catalyzes the first step, herein called the "deaminase," has been purified 200-fold. The activity was assayed by detecting the conversion of the product of the reaction catalyzed by GTP cyclohydrolase II to a compound which reacts with butanedione to form 6,7-dimethyllumazine. The enzyme has a molecular weight of approximately 80,000 and a pH optimum of 9.1. The dephosphorylated form of the substrate is not deaminated in the presence of the enzyme. The assay for the enzyme which catalyzes the second step, referred to here as the "reductase," involves the detection of the conversion of the product of the deaminase-catalyzed reaction to a compound which, after treatment with alkaline phosphatase, reacts with butanedione to form 6,7-dimethyl-8-ribityllumazine. The reductase has a molecular weight of approximately 40,000 and a pH optimum of 7.5. Like the deaminase, the reductase does not act on the dephosphorylated form of its substrate. Reduced nicotinamide adenine dinucleotide phosphate is required as a cofactor; reduced nicotinamide adenine dinucleotide can be used about 30% as well as the phosphate form. The activity of neither enzyme is inhibited by riboflavin, FMN, or flavine adenine dinucleotide.     

Detection of a tryptophan radical in the reaction of ascorbate peroxidase with hydrogen peroxide.

The reactivity of recombinant pea cytosolic ascorbate peroxidase (rAPX) towards H2O2, the nature of the intermediates and the products of the reaction have been examined using UV/visible and EPR spectroscopies together with HPLC. Compound I of rAPX, generated by reaction of rAPX with 1 molar equivalent of H2O2, contains a porphyrin pi-cation radical. This species is unstable and, in the absence of reducing substrate, decays within 60 s to a second species, compound I*, that has a UV/visible spectrum [lambda(max) (nm) = 414, 527, 558 and 350 (sh)] similar, but not identical, to those of both horseradish peroxidase compound II and cytochrome c peroxidase compound I. Small but systematic differences were observed in the UV/visible spectra of compound I* and authentic rAPX compound II, generated by reaction of rAPX with 1 molar equivalent H2O2 in the presence of 1 molar equivalent of ascorbate [lambda(max) (nm) = 416, 527, 554, 350 (sh) and 628 (sh)]. Compound I* decays to give a 'ferric-like' species (lambda(max) = 406 nm) that is not spectroscopically identical to ferric rAPX (lambda(max) = 403 nm) with a first order rate constant, k(decay)' = (2.7 +/- 0.3) x 10(-4) s(-1). Authentic samples of compound II evolve to ferric rAPX [k(decay) = (1.1 +/- 0.2) x 10(-3) s(-1)]. Low temperature (10 K) EPR spectra are consistent with the formation of a protein-based radical, with g values for compound I* (g parallel = 2.038, g perpendicular = 2.008) close to those previously reported for the Trp191 radical in cytochrome c peroxidase (g parallel = 2.037, g perpendicular = 2.005). The EPR spectrum of rAPX compound II was essentially silent in the g = 2 region. Tryptic digestion of the 'ferric-like' rAPX followed by RP-HPLC revealed a fragment with a new absorption peak near 330 nm, consistent with the formation of a hydroxylated tryptophan residue. The results show, for the first time, that rAPX can, under certain conditions, form a protein-based radical analogous to that found in cytochrome c peroxidase. The implications of these data are discussed in the wider context of both APX catalysis and radical formation and stability in haem peroxidases.     

Inhibition of the bovine branched-chain 2-oxo acid dehydrogenase complex and its kinase by arylidenepyruvates

A novel class of inhibitors for the branched-chain 2-oxo acid dehydrogenase (BCOAD) complex has been synthesized and studied. The sodium salts of arylidenepyruvates: e.g., furfurylidenepyruvate (compound I), 4-(3-thienyl)-2-oxo-3-butenoate (compound II), cinnamalpyruvate (compound III) and 4-(2-thienyl)-2-oxo-3-butenoate (compound IV) inhibit the overall and kinase reactions of the BCOAD complex from bovine liver. Inhibitions of the overall reaction occur at the decarboxylase (E1) step as determined by a spectrophotometric assay with 2,6-dichlorophenolindophenol as an electron acceptor. Inhibition of the E1 reaction by compound I (Ki = 0.5 microM) is competitive, whereas inhibitions by compounds II (Ki = 150 microM) and III (Ki = 500 microM) are non-competitive with respect to the substrate 2-oxoisovalerate. The Km value for 2-oxoisovalerate is 6.7 microM as measured by the E1 assay. Inhibition of the E1 step by compounds I, II and III are reversible at low inhibitor concentrations based on the Michaelis-Menten kinetics observed. By comparison, compound I does not significantly inhibit pyruvate and 2-oxoglutarate dehydrogenase complexes. The arylidenepyruvates (compounds I, II and IV) inhibit the BCOAD kinase reaction in a manner similar to the substrate 2-oxo acids. The inhibition of the kinase reaction by compound I is non-competitive with respect to ATP, with an apparent Ki value of 4.5 mM. The results suggest that arylidenepyruvates may be useful probes for elucidating the reaction mechanisms of the BCOAD complex and its kinase.     

Antioxidant activity of 5-alkoxymethyl-6-chromanols

The 5-alkoxymethyl-2,2,7,8-tetramethyl-6-chromanols (II) are excellent antioxidants against autoxidising safflower oil (ASO), although not as good as 2,2,5,7,8-pentamethyl-6-chromanol (I), the model compound of alpha-tocopherol. The aim of this work was to determine whether the rate of reaction of (II) with the radicals diphenylpicrylhydrazyl (DPP*) and galvinoxyl (ArO*) was directly proportional to their antioxidant activity against ASO. Compounds (II) reacted faster with DPP* than with ArO* but, in each case, slower than compound (I). The rates of reaction of I and II with both radicals followed the order I > II (R = H) > II (R = CH3) > II (R = other alkyls) and were directly proportional to their antioxidant activity against ASO.     

Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 degrees C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I-NADH reaction can be explained in terms of a single ionization of pKa = 4.7 +/- 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-II-NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 +/- 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II-NADH reaction was observed. Over the pH range of 4-10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 X 10(5) to 5.6 X 10(2) M-1 s-1 and of HRP-II with NADH varied from 4.4 X 10(4) to 4.1 M-1 s-1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.     

Synthesis of 17beta-N-substituted 19-Nor-10-azasteroids as inhibitors of human 5alpha-reductases I and II

The synthesis of 17beta-[N-(phenyl)methyl/phenyl-amido] substituted 10-azasteroids has been accomplished by either the TiCl4- or TMSOTf-catalysed reaction of carbamates 11 and 12 with Danishefsky's diene. The reaction provided 5alpha-H isomers 3a-5a and 5beta-H isomers 3b-5b depending on the reaction conditions. Both epimers of each compound were tested against human 5alpha-reductase types I and II. Unexpectedly, 5beta-H compounds were found more active than their 5alpha-H counterparts, the best inhibitors being 3b (IC50=279 and 2000 nM toward isoenzyme I and II, respectively) and 5b (IC50=913 and 247 nM toward isoenzymes I and II, respectively).     

Synthesis and characterization of cobalt and nickel chelates of 5-dimethylaminomethyl-2-thiouracil and their evaluation as antimicrobial and anticancer agents

A new antimetabolite of adenine, viz. 5-dimethylaminomethyl-2-thiouracil, was synthesized using the Mannich reaction. Owing to the biological importance of metalloelements in many biological processes, especially metabolic processes, cobalt(II) and nickel(II) complexes were also synthesized and examined for their antimicrobial and anticancer activities. These new compounds were characterized structurally by various techniques ranging from micro-elemental analyses to spectral analyses. Cobalt(II) complexes were found to be four coordinate, among which the bromo, iodo, and nitrato complexes were polymeric. The nickel(II) isothiocyanato complex exhibited four-coordinate geometry and the remaining nickel(II) complexes were six coordinate. Thermodynamic and kinetic parameters evaluated based on TG/DSC suggested that the initial stage of thermal decomposition follows a diffusion-controlled mechanism and the final stage a chemically controlled mechanism. Antibacterial, antifungal, and antitumor studies undertaken for the above compounds indicated structure-activity relationships. These metalloderivatives were more active than the parent compound. The order of activity was influenced by the chelate geometry and thermal stability. Activity increased with a decrease in coordination number and increase in thermal lability.     

Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2

The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 degree C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1 = 1.4 x 10(7) M-1 s-1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+.FeIVO]. Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2 = 65 s-1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome-c peroxidase, was assigned to a two-electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+. which was formed by an intramolecular electron transfer from tyrosine to the porphyrin-pi-cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.     

Reaction of ferrous cytochrome c peroxidase with dioxygen: site-directed mutagenesis provides evidence for rapid reduction of dioxygen by intramolecular electron transfer from the compound I radical site.

The reaction of dioxygen with the ferrous forms of the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was studied by photolysis of the respective ferrous-CO complexes in the presence of dioxygen. Reaction of ferrous CCP(MI) with dioxygen transiently formed a FeII-O2 complex (bimolecular rate constant = (3.8 +/- 0.3) x 10(4) M-1 s-1 at pH 6.0; 23 degrees C) that reacted further (first-order rate constant = 4 +/- 1 s-1) to form a product with an absorption spectrum and an EPR radical signal at g = 2.00 that were identical to those of compound I formed by the reaction of CCP(MI)III with peroxide. Thus, the product of the reaction of CCP(MI)II with dioxygen retained three of the four oxidizing equivalents of dioxygen. Gel electrophoresis of the CCP(MI)II + dioxygen reaction products showed that covalent dimeric and trimeric forms of CCP(MI) were produced by the reaction of CCP(MI)II with dioxygen. Photolysis of the CCP(MI)II-CO complex in the presence of ferrous cytochrome c prevented the appearance of the cross-linked forms and resulted in the oxidation of 3 mol of cytochrome c/mol of CCP(MI)II-CO added. The results provide evidence that reaction of CCP(MI)II with dioxygen causes transient oxidation of the enzyme by 1 equiv above the normal compound I oxidation state. Mutations that eliminate the broad EPR signal at g = 2.00 characteristic of the compound I radical also prevented the rapid oxidation of the ferrous enzyme by dioxygen.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of V(III)-adenine complex on yeast as a model of eukaryotic cells

The dinuclear micro-okso vanadium (III) complex compound H(4)V(2)OCl(4)(Ad)(2) synthesized in our laboratory was investigated as a potential cytotoxic agent against yeast cells. The results of these studies could be helpful in the explanation of the mechanism governing the V (III) compound action on yeast as a simple model of eukaryotic cells. The important factors influencing the toxicity of this complex compound are: the stage of the yeast life cycle, the rate of growth and the pH of reaction mixture. The lethal effect was distinctly stronger when the reaction mixture was slightly acidic (pH = 4). In such solutions V(III) mononuclear species with adenine was relatively stable, and during the time of experiment possibly only a slow oxidation process to V(IV) occurred. In the solutions with pH = 7, several hydrolytic, perhaps mixed-valence, polynuclear species were present and their action on the yeast cells was rather weak. The increased lethal activity of this compound in acidic solutions may be useful in specific treatment against cancer cells whose cytoplasm and/or closest surrounding has lower pH value. The next important result was an observation that the killing activity of this compound was enhanced for yeast cells being in log phase. Also these which had a slower rate of growth (possessing some auxotrophic mutations) were more resistant than those growing faster. The extent of yeast mutagenesis caused by the complex compound is negligible, as the number of mutants found in experiments was within the limit of experimental error. These results are promising and the investigated complex can be considered as a potential anti cancer agent.     

Oxidation of aromatic sulfides by lignin peroxidase from Phanerochaete chrysosporium.

The reaction of H2O2 with 4-substituted aryl alkyl sulfides (4-XC6H4SR), catalysed by lignin peroxidase (LiP) from Phanerochaete chrysosporium, leads to the formation of sulfoxides, accompanied by diaryl disulfides. The yields of sulfoxide are greater than 95% when X = OMe, but decrease significantly as the electron donating power of the substituent decreases. No reaction is observed for X = CN. The bulkiness of the R group has very little influence on the efficiency of the reaction, except for R = tBu. The reaction exhibits enantioselectivity (up to 62% enantiomeric excess with X = Br, with preferential formation of the sulfoxide with S configuration). Enantioselectivity decreases with increasing electron density of the sulfide. Experiments in H218O show partial or no incorporation of the labelled oxygen into the sulfoxide, with the extent of incorporation decreasing as the ring substituents become more electron-withdrawing. On the basis of these results, it is suggested that LiP compound I (formed by reaction between the native enzyme and H2O2), reacts with the sulfide to form a sulfide radical cation and LiP compound II. The radical cation is then converted to sulfoxide either by reaction with the medium or by a reaction with compound II, the competition between these two pathways depending on the stability of the radical cation.     

Catalytic mechanism and three-dimensional structure of adenine deaminase

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k(cat) = 2.0 s(-1); k(cat)/K(m) = 2.5 × 10(3) M(-1) s(-1)). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn(2+) prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k(cat) and k(cat)/K(m) values of 200 s(-1) and 5 × 10(5) M(-1) s(-1), respectively. The apoenzyme was prepared and reconstituted with Fe(2+), Zn(2+), or Mn(2+). In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe(II)/Fe(II)]-ADE was oxidized to [Fe(III)/Fe(III)]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe(III)/Fe(III)]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Mo?ssbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 ? resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate-limiting steps.     

The redox properties of ascorbate peroxidase

Reduction potentials for the catalytic compound I/compound II and compound II/Fe3+ redox couples, and for the two-electron compound I/Fe3+ redox couple, have been determined for ascorbate peroxidase (APX) and for a number of site-directed variants. For the wild type enzyme, the values are E degrees '(compound I/compound II) = 1156 mV, E degrees '(compound II/Fe3+) = 752 mV, and E degrees '(compound I/Fe3+) = 954 mV. For the variants, the analysis also includes determination of Fe3+/Fe2+ potentials which were used to calculate (experimentally inaccessible) E degrees '(compound II/Fe3+) potentials. The data provide a number of new insights into APX catalysis. The measured values for E degrees '(compound I/compound II) and E degrees '(compound II/Fe3+) for the wild type protein account for the much higher oxidative reactivity of compound I compared to compound II, and this correlation holds for a number of other active site and substrate binding variants of APX. The high reduction potential for compound I also accounts for the known thermodynamic instability of this intermediate, and it is proposed that this instability can account for the deviations from standard Michaelis kinetics observed for most APX enzymes during steady-state oxidation of ascorbate. This study provides the first systematic evaluation of the redox properties of any ascorbate peroxidase using a number of methods, and the data provide an experimental and theoretical framework for accurate determination of the redox properties of Fe3+, compound I, and compound II species in related enzymes.     

Resonance Raman spectra of bovine liver catalase compound II. Similarity of the heme environment to horseradish peroxidase compound II

Resonance Raman spectroscopy has been used to investigate the structure and environment of the heme group in bovine liver catalase compound II. Both Soret- and Q-band excitation have been employed to observe and assign the skeletal stretching frequencies of the porphyrin ring. The oxidation state marker band v4 increases in frequency from 1373 cm-1 in ferricatalase to 1375 cm-1 in compound II, consistent with oxidation of the iron atom to the Fe(IV) state. Oxidation of five-coordinate, high-spin ferricatalase to compound II is accompanied by a marked increase of the porphyrin core marker frequencies that is consistent with a six-coordinate low-spin state with a contracted core. An Fe(IV) = O stretching band is observed at 775 cm-1 for compound II at neutral pH, indicating that there is an oxo ligand at the sixth site. At alkaline pH, the Fe(IV) = O stretching band shifts to 786 cm-1 in response to a heme-linked ionization that is attributed to the distal His-74 residue. Experiments carried out in H218O show that the oxo ligand of compound II exchanges with bulk water at neutral pH, but not at alkaline pH. This is essentially the same behavior exhibited by horseradish peroxidase compound II and the exchange reaction at neutral pH for both enzymes is attributed to acid/base catalysis by a distal His residue that is believed to be hydrogen-bonded to the oxo ligand. Thus, the structure and environment of the heme group of the compound II species of catalase and horseradish peroxidase are very similar. This indicates that the marked differences in their reactivities as oxidants are probably due to the manner in which the protein controls access of substrates to the heme group.     

Characterization of fluorescent products from reaction of methyl linoleate hydroperoxides with adenine in the presence of Fe2+ and ascorbic acid

The structures of fluorescent products formed in the reaction of methyl linoleate hydroperoxides with adenine, FeSO4 and ascorbic acid were investigated to elucidate the mechanism of interaction. The fluorescent products consisted of at least four major components (I-IV), which could be separated by thin-layer chromatography and high-performance liquid chromatography. Both 2-octenal and 2,4-decadienal, degradation products of methyl linoleate hydroperoxides, reacted with adenine to produce a fluorescent product similar to one of the major compounds (II) formed in the reaction of methyl linoleate hydroperoxides. Spectroscopic data suggest that I and III are the same type of compounds, which have closed ring structures with alpha, beta-unsaturated carbonyl groups between the amino group at the 6-position and the nitrogen at the 1-position of adenine. Component II has a closed ring structure at the same site as I and III, and the presence of an ether linkage was suggested. On the basis of these structures, the involvement of 3-nonenal, methyl 12-oxo-9-dodecenoate and 2-octenal was suggested in the interaction of the methyl linoleate hydroperoxides decomposition products and adenine or DNA in the presence of FeSO4 and ascorbic acid.     

Stoichiometry of the reaction between horseradish peroxidase and p-cresol.

Over a wide range of pH horseradish peroxidase compound I can be reduced quantitatively via compound II to the native enzyme by only 1 molar equivalent of p-cresol. Since 2 molar equivalents of electrons are required for the single turnover of the enzymatic cycle, p-cresol behaves as a 2-electron reductant. With p-cresol and compound I in a 1:1 ratio compound II and p-methylphenoxy radicals are obtained in the transient state. Compound II is then reduced to the native enzyme. A possible explanation for the facile reduction of compound II involves reaction with the dimerization product of these radicals, 1/2 molar equivalent of 2,2'-dihydroxy-5,5'-dimethylbiphenyl. If only 1/2 molar equivalent of p-cresol is present, than at high pH the reduction stops at compound II. The major steady state peroxidase oxidation product of p-cresol (with p-cresol in large excess compared to the enzyme concentration) is Pummerer's ketone. Pummerer's ketone is only reactive at pH values greater than about 9 where significant amounts of the enol can be formed via the enolate anion. Therefore, in alkaline solution it is reactive with compound I, but not with compound II, which is converted into an unreactive basic form. These results indicate that Pummerer's ketone cannot be the intermediate free radical product responsible for reducing compound II in the single turnover experiments. It is postulated that Pummerer's ketone is formed only in the steady state by the reaction of the p-methylphenoxy radical with excess p-cresol.     

The Reaction Rates of NO with Horseradish Peroxidase Compounds I and II

In this study the reactions between nitric oxide (NO) and horseradish peroxidase (HRP) compounds I and II were investigated. The reaction between compound I and NO has biphasic kinetics with a clearly dominant initial fast phase and an apparent second-order rate constant of (7.0 +/- 0.3) x 10(5) M(-1) s(-1) for the fast phase. The reaction of compound II and NO was found to have an apparent second-order rate constant of k(app) = (1.3 +/- 0.1) x 10(6) M(-1) s(-1) or (7.4 +/- 0.7) x 10(5) M(-1) s(-1) when measured at 409 nm (the isosbestic point between HRP and HRP-NO) and 419 nm (lambda(max) of compound II and HRP-NO), respectively. Interestingly, the reaction of compound II with NO is unusually high relative to that of compound I, which is usually the much faster reaction. Since horseradish peroxidase is prototypical of mammalian peroxidases with respect to the oxidation of small substrates, these results may have important implications regarding the lifetime and biochemistry of NO in vivo after inflammation where both NO and H(2)O(2) generation are increased several fold.     

Enzymatic Synthesis of l-Carnitine by Reduction of an Achiral Precursor: the Problem of Reduced Nicotinamide Adenine Dinucleotide Recycling

Synthesis of l-carnitine has been carried out by the enzymatic reduction of the carbonyl group of the achiral precursor 3-dehydrocarnitine with the oxidized nicotinamide adenine dinucleotide-linked carnitine dehydrogenase. Various enzymatic or chemical systems have been tested to regenerate the reduced nicotinamide adenine dinucleotide oxidized in the reduction of 3-dehydrocarnitine. Because of the instability of this compound in aqueous solutions, it was added by continuous feeding as a rate-limiting constituent in the reaction mixture. Under these conditions, conversion yields of 95% were achieved with the glucose plus glucose dehydrogenase system. A total number of 530 reduced nicotinamide adenine dinucleotide recyclings was obtained with this system for a production of 45 g of l-carnitine per liter. The stabilities of the oxidized nicotinamide adenine dinucleotide and the reduced nicotinamide adenine dinucleotide have been determined at various pH values. In view of these results, several possible strategies for enzymatic syntheses with the reduced nicotinamide adenine dinucleotide as a regenerable coenzyme are discussed.