The One-Electron Reduction Potential of 3-Amino-1, 2, 4-benzotriazine 1, 4-dioxide (Tirapazamine): A Hypoxia-Selective Bioreductive Drug

The one-electron reduction potential of 3-amino-l, 2, 4-benzotriazine 1, 4-dioxide, tirapazamine (SR 4233) in aqueous solution has been determined by pulse radiol-ysis. Reversible electron transfer was achieved between radiolytically-generated one-electron reduced radicals of tirapazamine (T), and quinones or benzyl viologen as redox standards. The reduction potential E_m7(T/T^±) was -0.45 ± 0.01 V vs. NHE at pH 7. From the pH dependence of the reduction potential, pK_a = 5.6 ± 0.2 was estimated for the tirapazamine radical, a value similar to the pK_a determined by other methods.     

Chemically accurate protein structures: Validation of protein NMR structures by comparison of measured and predicted pK a values

A new method is presented for evaluating the quality of protein structures obtained by NMR. This method exploits the dependence between measurable chemical properties of a protein, namely pK _a values of acidic residues, and protein structure. The accurate and fast empirical computational method employed by the PROPKA program () allows the user to test the ability of a given structure to reproduce known pK _a values, which in turn can be used as a criterion for the selection of more accurate structures. We demonstrate the feasibility of this novel idea for a series of proteins for which both␣NMR and X-ray structures, as well as pK _a values of all ionizable residues, have been determined. For the 17 NMR ensembles used in this study, this criterion is shown effective in the elimination of a large number of NMR structure ensemble members.     

Cytochrome b5 reductase–cytochrome b5 as an active P450 redox enzyme system in Phanerochaete chrysosporium: Atypical properties and in vivo evidence of electron transfer capability to CYP63A2

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b₅ reductase–cyt b₅. In fungi, limited information is available for the cyt b₅ reductase–cyt b₅ system. Here we characterized the kinetic mechanism of (cyt b₅r)–cyt b₅ redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b₅r followed a “ping-pong” mechanism and could directly reduce cytochrome c. However, unlike other cyt b₅ reductases, Pc-cyt b₅r lacked the typical ferricyanide reduction activity, a standard for cyt b₅ reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b₅r–cyt b₅ complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b₅r–cyt b₅ complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b₅r–cyt b₅) in terms of supporting a P450 monooxygenase activity.     

Synthesis and binding assays of novel 3,3-dimethylpiperidine derivatives with various lipophilicities as σ1 receptor ligands

Starting from two carbocyclic analogs, a series of 3,3-dimethylpiperidine derivatives was prepared and tested in radioligand binding assays at σ₁ and σ₂ receptors, and at Δ₈–Δ₇ sterol isomerase (SI) site. The novel compounds mostly bear heterocyclic rings or bicyclic nucleus of differing lipophilicities. Compounds 18a and 19a,b demonstrated the highest σ₁ affinity (K_i = 0.14–0.38 nM) with a good selectivity versus σ₂ binding. Among them, 18a had the lowest C log D value (3.01) and only 19b was selective versus SI too. Generally, it was observed that more planar and hydrophilic heteronuclei conferred a decrease in affinity for both σ receptor subtypes.     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

Effects of temperature and CO2 enrichment on kinetic properties of NADP+-malate dehydrogenase in two ecotypes of Barnyard grass (Echinochloa crus-galli (L.) Beauv.) from contrasting climates

Summary The apparent energy of activation (E _a), Michaelis-Menten constant (K _mfor oxaloacetate), V _max/K _mratios and specific activities of NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) were analyzed in plants of Barnyard grass from Québec (QUE) and Mississippi (MISS) acclimated to two thermoperiods 28/22°C, 21/15°C, and grown under two CO₂ concentrations, 350 l l^-1 and 675 l l^-1. E _avalues of NADP⁺-MDH extracted from QUE plants were significantly lower than those of MISS plants. K _mvalues and V _max/K _mratios of the enzyme from both ecotypes were similar over the range of 10–30°C but reduced V _max/K _mratios were found for the enzyme of QUE plants at 30 and 40°C assays. MISS plants had higher enzyme activities when measured on a chlorophyll basis but this trend was reversed when activities were expressed per fresh weight leaf or per leaf surface area. Activities were significantly higher in plants of both populations acclimated to 22/28°C. CO₂ enrichment did not modify appreciably the catalytic properties of NADP⁺-MDH and did not have a compensatory effect upon catalysis or enzyme activity under cool acclimatory conditions. NADP⁺-MDH activities were always in excess of the amount required to support observed rates of CO₂ assimilation and these two parameters were significantly correlated. The enhanced photosynthetic performance of QUE plants under cold temperature conditions, as compared to that of MISS plants, cannot be attributed to kinetic differences of NADP⁺-malate dehydrogenase among these ecotypes.     

A novel low-temperature active alkaline pectate lyase from Klebsiella sp. Y1 with potential in textile industry

Alkaline pectate lyases are favorable for the textile industry. Here we report the cloning of a pectate lyase gene (pl A), from Klebsiella sp. Y1, and its heterologous expression in Escherichia coli. The full-length pl A consists of 1710 bp and encodes for a 569-amino acid polypeptide including a putative 22-residue signal peptide and a catalytic domain belonging to pectate lyase family 2. The recombinant enzyme (r-PL A) was purified to electrophoretic homogeneity by single-step Ni²⁺-NTA affinity chromatography and showed an apparent molecular weight of ∼60 kDa. The pH and temperature optima of r-PL A were found to be 9.0 and 30–50 °C, respectively. r-PL A was highly active at low temperatures, exhibiting >60% of the maximal activity at 20 °C and >20% activity even at 0 °C. The enzyme was stable in a broad alkaline pH range of 7.0–12.0 for 1 h at 37 °C. The values of K_m(app) and V_max(app) of r-PL A for polygalacturonic acid were 2.47 mg/ml and 11.94 μmol/min/mg, respectively. Compared with the commercial compound pectinase from Novozymes, purified r-PL A showed similar efficacy in reducing the intrinsic viscosity of polygalacturonic acid (68.8% vs. 67.1%) and in bioscouring of jute (7.38% vs. 7.58%). Thus r-PL A is a valuable material for the textile industry.     

Purification and characterization of a new type of serine carboxypeptidase from<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K_m, V_max, K_cat, and K_cat/K_m values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min^–1, 291 s^–1, and 339 mM^–1 s^–1, respectively.     

Acetylcholinesterase/Butyrylcholinesterase inhibition activity of some new carbacylamidophosphate deriviatives

Eight newly synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl₂ with R = pCl–C₆H₄ 1a, pBr–C₆H₄ 2a, C₆H₅ 3a, and pMe–C₆H₄ 4a and RC(O)NHP(O)(NC₄H₈O)₂ R = pCl–C₆H₄ 1b, pBr–C₆H₄ 2b, C₆H₅ 3b, pMe–C₆H₄ 4b, were selected to compare the inhibition kinetic parameters, IC₅₀, K_i, k_p and K_D, on human erythrocyte acetylcholinesterase (hAChE) and bovine serum butyrylcholinesterase (BuChE), Also, the in vivo inhibition potency of compound 2a, 2b and 3a, were studied. The data demonstrates that compound 2a and compound 2b are the potent sensitive as AChE and BuChE inhibitors respectively, and the inhibition of hAChE is about 10-fold greater than that of BuChE.     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of -D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V ₁/K _B , for reactions of these substrates were collected from pH 2.5–8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of -D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid.Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pK _a of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pK _a of His516 in the free reduced enzyme is 6.9.     

Relevance of phenylalanine 216 in the affinity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phosphoenolpyruvate carboxykinase for Mn(II)

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn²⁺ as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. (1997) Nature Struct. Biol. 4, 990–994] shows that Lys²¹³ is one of the ligands to Mn²⁺ at the enzyme active site. Coordination of Mn²⁺ to a lysyl residue is not common and suggests a low pK _a value for the ε-NH₂ group of Lys²¹³. In this work, we evaluate the role of neighboring Phe²¹⁶ in contributing to provide a low polarity microenvironment suitable to keep the ε-NH₂ of Lys²¹³ in the unprotonated form. Mutation Phe216Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn²⁺, suggesting an increase of the pK _a of Lys²¹³. In agreement with this interpretation, theoretical calculations indicate an alkaline shift of 2.8 pH units in the pK _a of the ε-amino group of Lys²¹³ upon Phe216Tyr mutation.     

An Arginine Specific Protease from Spirulina platensis

An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues, including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA) was hydrolyzed most effectively with the enzyme (K_m = 5.5 × 10⁻⁶ M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited completely with camostat mesilate (K_i = 1.1 × 10⁻⁸ M) and leupeptin (K_i = 3.9 × 10⁻⁸ M) but was not inhibited with N^α-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature was 50°C; the enzyme was stable at 0–50°C.     

Synthesis and antileishmanial evaluation of 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazole derivatives

A series of 1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (4a–g) and 5-amino-1-aryl-4-(4,5-dihydro-1H-imidazol-2-yl)-1H-pyrazoles (5a–g) were synthesized and evaluated in vitro against three Leishmania species: L. amazonensis, L. braziliensis and L. infantum (L. chagasi syn.). The cytotoxicity was assessed. Among the derivatives examined, six compounds emerged as the most active on promastigotes forms of L. amazonensis with IC₅₀ values ranging from 15 to 60 μM. The reference drug pentamidine presented IC₅₀ = 10 μM. However, these new compounds were less cytotoxic than pentamidine. Based on these results, the more promising derivative 5d was tested further in vivo. This compound showed inhibition of the progression of cutaneous lesions in CBA mice infected with L. amazonensis relative to an untreated control.     

Properties of a novel β-glucosidase from Fusarium proliferatum ECU2042 that converts ginsenoside Rg3 into Rh2

A novel β-glucosidase from Fusarium proliferatum ECU2042 (FPG) was successfully purified to homogeneity with a 506-fold increase in specific activity. The molecular mass of the native purified enzyme (FPG) was estimated to be approximately 78.7 kDa, with two homogeneous subunits of 39.1 kDa, and the pI of this enzyme was 4.4, as measured by two-dimensional electrophoresis. The optimal activities of FPG occurred at pH 5.0 and 50 °C, respectively. The enzyme was stable at pH 4.0–6.5 and temperatures below 60 °C, and the deactivation energy (E_d) for FPG was 88.6 kJ mo1⁻¹. Moreover, it was interesting to find that although the purified enzyme exhibited a very low activity towards p-nitrophenyl β-d-glucoside (pNPG), and almost no activity towards cellobiose, a relatively high activity was observed on ginsenoside Rg₃. The enzyme hydrolyzed the 3-C, β-(1 → 2)-glucoside of ginsenoside Rg₃ to produce ginsenoside Rh₂, but did not sequentially hydrolyze the β-d-glucosidic bond of Rh₂. The K_m and V_max values of FPG for ginsenoside Rg₃ were 2.37 mM and 0.568 μmol (h mg protein)⁻¹, respectively. In addition, this enzyme also exhibited significant activities towards various alkyl glucosides, aryl glucosides and several natural glycosides.     

Isolation and Study of Some Properties of Laccase from the Basidiomycetes Cerrena maxima

A new strain producing extracellular laccase (Cerrena maxima 0275) was found by screening of isolates of Basidiomycetes, and the dynamics of laccase biosynthesis by this strain was studied. The enzyme was purified to homogeneity. The molecular weight of the enzyme is 57 kD, and its pI is 3.5. The activity is constant at pH values in the range 3.0-5.0. The temperature optimum for activity is 50°C. The thermal stability of the laccase was studied. The catalytic and Michaelis constants for catechol, hydroquinone, sinapinic acid, and K₄ Fe(CN)₆ were determined. The standard redox potential of type 1 copper in the enzyme is 750 ± 5 mV. Thus, the investigated laccase is a high redox potential laccase.     

The role of intramolecular hydrogen bonding on nucleobase acidification following metal coordination: possible implications of an “indirect” role of metals in acid–base catalysis of nucleic acids

The acidifying effect of Pt^II on nucleobase –NH and –NH₂ groups depends both on the site of metal coordination and on the efficiency of stabilization of the deprotonated nucleobase via intracomplex hydrogen bonding. Weakly acidic nucleobase protons with pK _a values between 9 and 17 can be acidified by a single Pt^II to have pK _a values which are well within the physiological pH range. This could open the possibility of an acid–base catalysis occurring at pH 7, with the metal–nucleobase entity functioning either as an acid or a base. Examples of Pt^II complexes studied here include, among others, mixed nucleobase systems of 1-methylcytosine and 1,9-dimethyladenine as well as a complex of the rare iminooxo tautomer of 1-methylcytosine having the metal bonded at N4.     

Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase

Improvement of enzyme function by engineering pH dependence of enzymatic activity is of importance for industrial application of Bacillus circulans xylanases. Target mutation sites were selected by structural alignment between B. circulans xylanase and other xylanases having different pH optima. We selected non-conserved mutant sites within 8 Å from the catalytic residues, to see whether these residues have some role in modulating pK_as of the catalytic residues. We hypothesized that the non-conserved residues which may not have any role in enzyme catalysis might perturb pK_as of the catalytic residues. Change in pK_a of a titratable group due to change in electrostatic potential of a mutation was calculated and the change in pH optimum was predicted from the change in pK_a of the catalytic residues. Our strategy is proved to be useful in selection of promising mutants to shift the pH optimum of the xylanases towards desired side.     

Cathepsins B, H and L in Peritoneal Macrophages and Hepatopancreas of Carp Cyprinus carpio

Cathepsin B was purified from the crude extract of carp hepatopancreas by a modified method, and the specific activity increased about 3,400-fold with a 17% recovery. The purified enzyme gave a single protein band on Native-PAGE, whereas two bands with molecular weights of 30,000 (single chain) and 26,000 (heavy chain) migrated on SDS-PAGE. The enzyme potently hydrolyzed Z-Arg-Arg-MCA and Z-Phe-Arg-MCA but lacked the ability to hydrolyze most of the other MCA substrates. The kinetic constants of the enzyme with two Z-blocked substrates revealed that V_max and K_cat values of Z-Phe-Arg-MCA were much higher than Z-Arg-Arg-MCA, while their K_m values were approximate. The optimum hydrolysis pH and temperature of the enzyme for these two substrates were determined to be pH 6.0 and 45°C, respectively. A variety of protease inhibitors such as E-64, DTNB, antipain, leupeptin, chymostatin, TLCK and TPCK and metal compounds such as CuCl₂, CdCl₂, HgCl₂, and Zn(CH₃COO)₂ were able to significantly inactivate the enzyme. In contrast, cysteine and 2-mercaptoethanol activated the enzyme, with cysteine being more effective for activation than 2-mercaptoethanol over the tested concentrations.     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses; therefore, the knowledge of the volumetric mass transfer coefficient (k_La) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k_La values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium, in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2² full factorial design. Both variables showed statistically significant effects on k_La, and the highest values of this parameter in both media for simple fermentation (241 s⁻¹) and extractive fermentation with ATPS (70.3 s⁻¹) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k_La values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N³D²) and superficial gas velocity (V_s) determined in distilled water (α = 0.39 and β = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (α = 0.38 and β = 0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (α = 0.50 and β = 1.0). A reasonable agreement was found between the experimental data of k_La for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions.