Stabilities and rates in the laccase/TEMPO-catalyzed oxidation of alcohols

The influence of alcohol, 4-acetylamino,2,2,6,6'-tetramethylpiperidinyloxy (4-acetylamino-TEMPO) and laccase (from Trametes versicolor, TvL) concentration in the aerobic oxidation of furfuryl alcohol was investigated. Studies show that the K _m for 4-acetylamino-TEMPO is around 6.3 mM (V _max=0.18 mM min^-1) using 6.6 U mL^-1 of laccase and a furfuryl alcohol concentration of 140 mM. Under these optimized conditions, the reaction rate is still dependent on the concentration of enzyme in solution. Laccase can be reused, with a residual activity of around 25%. An important conclusion is that laccase is not stable in the presence of oxoammonium salts, presumably due to degradation via oxidation of essential amino acid residues or the glycosyl moieties on the periphery of the enzyme.     

Carboxypeptidase Y-Catalyzed Reaction in Systems Containing Organic Solvent

The effect of organic solvents on carboxypeptidase Y (a serine carboxypeptidase from yeast)-catalyzed hydrolysis of amino acid ester and peptide synthesis from N-acyl amino acid ester and amino acid amide was investigated.

The K_m value of ester hydrolysis increased with an increase in the solvent content. Dioxane was the most effective and dimethyl sulfoxide (DMSO) the least, whilst K_cat showed a tendency to increase slightly in N, N-dimethylformamide (DMF) and DMSO. For dioxane and acetonitrile (MeCN) a maximum was observed.

In peptide formation from Fua-Phe-OEt and Gly-NH₂, dioxane and MeCN supported high product yield at molar fractions smaller than ca. 0.05 but the yield decreased significantly at higher fractions, although a relatively constant selectivity (ratio of the peptide bond formed to the ester consumed) was maintained. DMSO gave rather low peptide yields and selectivity even at lower molar fractions. DMF showed an intermediate tendency.

An apparent saturation parameter of the amine component was evaluated and the dissociation constant of a complex between acyl-enzyme and amino acid amide (K_n), as well as the rate constant of aminolysis exerted by the amino acid amide bound correctly on the enzyme (K_n), was calculated by initial rate analysis of peptide formation. In contrast to K_m values, K_n decreased with increasing concentrations of organic cosolvent. while a suppressive effect was observed (except for DMSO) on the K_n parameter.

Effects of the solvent practically immiscible in water was also studied by use of the enzyme physically “immobilized” on glass beads.     

Energy transduction in photosynthetic bacteria IV. Light-dependent ATPase in photosynthetic membranes from Rhodopseudomonas capsulata

ATPase activity of photosynthetic membrane fragments from the bacterium Rhodopseudomonas capsulata can be stimulated by continuous illumination under conditions of active cyclic electron flow. The activation corresponds to an increase in the maximum velocity of the reaction and does not affect the apparent K_m for ATP (0.11 mM). No stimulation in the light is observed in the presence of classical uncouplers or oxidized 2,6-dichlorophenolindophenol (DCIP), which, per se, stimulate ATPase in the dark. It is demonstrated, however, that oxidized DCIP acts as an uncoupler of bacterial photophosphorylation.

The effect of light is elicited after a few minutes of preillumination, or in a much shorter time if an ADP trapping system is supplied. Activation does not occur if ADP is added during the preillumination (apparent K_m for inhibition by ADP = 1 μM). The effect of ADP is not related to competitive inhibition with ATP, which can be observed at higher concentrations (apparent K_i = 0.26 mM). ADP, however, is not effective if added after some minutes of preillumination.     

Nonionic detergent-induced activation of an esterase from Bacillus megaterium 20-1

An esterase-producing Bacillus megaterium strain (20-1) was isolated from a soil sample collected in South Korea. The cloned gene showed that the esterase 20-1 composed of 310 amino acids corresponding to a molecular mass (M_r) of 34,638. Based on the M_r and the protein sequence, the esterase 20-1 belonged to the H lipase/esterase group. The optimum temperature and pH of the purified His-tagged enzyme were 20–35 °C and 8.0, respectively. The esterase 20-1 showed a ‘nonionic detergent-induced activation’ phenomenon, which was a detergent type- and concentration-dependent process. In comparison with the native enzyme, the Tween 80-treated enzyme had relatively a similar k_cat value of 274 s⁻¹ but a very low K_m value of 0.037 mM for PNPC (C₆), therefore, it showed a 14-fold increase in k_cat/K_m value.     

Biochemical and biophysical studies on cytochrome c oxidase XV. Reaction with fluoride

1. Fluoride is a mixed-type inhibitor of the cytochrome c oxidase activity with a K_i for the free enzyme of 10 mM and a K_i for the cytochrome c-complexed enzyme of 35 mM.

2. Fluoride shifts the γ-band of the enzyme from 423 to 421 nm and the -band from 597 to 598 nm. The difference spectrum (oxidized enzyme in the presence of fluoride minus oxidized enzyme) has peaks at 400, 453, 482, 605 and 638 nm and troughs at 430, 520, 552 and 674 nm. The changes in absorbance are small (about 3% at absorbance maxima) with respect to those of other hemoproteins.

3. On addition of fluoride to isolated cytochrome c oxidase 3 reactions can be distinguished: (I) a bimolecular binding reaction (K_on = 4 M⁻¹ · s⁻¹ and k_off = 2.9 · 10⁻²s⁻¹ at 25 °C, pH 7.4) contributing at 638 nm and 430 nm; (II) a first-order reaction (k = 2.4 · 10⁻²) s⁻¹ at 22 °C, pH 7.2) visible mainly at 430 nm and (III) a very slow reaction with a half-time in the order of 10 min.

4. The spectroscopic dissociation constants for the fluoride binding, determined from Hill plots using the absorbance changes at 638 and 430 nm, are similar (7 and 10 mM, respectively, at 22 °C, pH 7.2).

5. A mechanism for the reaction is discussed in which the bimolecular binding reaction is followed by a conformational change of the enzyme-fluoride complex.     

Modulation of ouabain-insensitive Na(+)-ATPase activity in the renal proximal tubule by Mg(2+), MgATP and furosemide

In addition to the (Na⁺+K⁺)ATPase another P-ATPase, the ouabain-insensitive Na⁺-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg²⁺, MgATP and furosemide on the Na⁺-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na⁺-ATPase at pH 7.0 are: (a) K_0.5 for Na⁺=8.9±2.2 mM; (b) K_0.5 for MgATP=1.8±0.4 mM; (c) two sites for free Mg²⁺: one stimulatory (K_0.5=0.20±0.06 mM) and other inhibitory (I_0.5=1.1±0.4 mM); and (d) I_0.5 for furosemide=1.1±0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na⁺ (K_0.5=19.5±0.4) and MgATP (K_0.5=3.4±0.3 mM) but increases the apparent affinity for furosemide (0.18±0.02 mM) and Mg²⁺ (0.05±0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na⁺ (K_0.5=18.7±1.5 mM) and furosemide (I_0.5=3.04±0.57 mM) but does not change the apparent affinity to MgATP and Mg²⁺. The data presented in this paper indicate that the modulation of the Na⁺-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg²⁺ and MgATP complex may play an important role in the Na⁺-ATPase physiological regulatory mechanisms.     

Thermostable and alkalophilic maltogenic amylase of Bacillus thermoalkalophilus ET2 in monomer-dimer equilibrium

A gene encoding a thermostable and alkalophilic maltogenic amylase (BTMA) was cloned from the thermophilic bacterium Bacillus thermoalkalophilus ET2. BTMA was composed of 588 amino acids with a predicted molecular mass of 68.8 kDa. The enzyme had an optimal temperature and pH of 70°C and 8, respectively, the highest among maltogenic amylases reported so far. The Tm of BTMA at pH 8 was 76.7°C with an enthalpy of 113.6 kJ mol^-1. Both hydrolysis and transglycosylation activities for various carbohydrates were evident. β-Cyclodextrin (β-CD) and soluble starch were hydrolyzed mainly to maltose, and pullulan to panose. Acarbose, a strong amylase inhibitor, was hydrolyzed by BTMA to glucose and acarviosine-glucose. The K _m and k _cat values of BTMA for β-CD hydrolysis were 0.128 mM and 165.8 s^-1 mM, respectively. The overall catalytic efficiency (k _cat/K _m) of the enzyme was highest toward β-CD. BTMA was present in a monomer-dimer equilibrium with a molar ratio of 54:46 in 50 mM glycine-NaOH buffer (pH 8.0). This equilibrium could be affected by KCl and enzyme concentrations. The multi-substrate specificity of the enzyme was modulated by the structural differences between monomeric and dimeric forms. Starch was hydrolyzed more readily when monomeric BTMA was prevalent, while the opposite was observed for β-CD.     

Characterization of reactions catalysed by yeast phosphatidylinositol synthase

The nature of reactions catalysed by yeast phosphatidylinositol synthase expressed in E. coli has been investigated. The single enzyme is shown to carry both CDP-diacylglycerol-dependent incorporation of inositol into phosphatidylinositol (K_m for inositol of 0.090 mM) and a CDP-diacylglycerol-independent exchange reaction between phosphatidylinositol and inositol (K_m for inositol of 0.066 mM). The exchange reaction and reversal of phosphatidylinositol synthase were both stimulated by CMP, but had different optimum pH and requirements for substrates. These results suggest that CMP-stimulated exchange and CMP-dependent reverse reactions are distinct processes catalysed by the same enzyme. phosphatidylinositol synthase.     

Mechanistic investigations of 1-aminocyclopropane 1-carboxylic acid oxidase with alternate cyclic and acyclic substrates

The behavior of three cyclic and three acyclic analogues of 1-aminocyclopropane-1-carboxylic acid (ACC) with ACC oxidase has been analyzed with regard to turnover rates, product distribution, and O(2) uncoupling. The cyclic analogues all form ethylene, and the acyclic analogues all undergo decarboxylation. The degree of uncoupling varies from almost none (ACC) to 21-fold (glycine), while turnover rates (k(cat)) are all within a factor of 4-fold of that of ACC. The aggregate data point toward a rate-determining formation of an activated iron-oxo intermediate, which partitions between amine oxidation and reductive uncoupling in a manner that is dependent on substrate structure.     

Immobilization of β-galactosidase from Kluyveromyces lactis on silica and agarose: comparison of different methods

The covalent immobilization of β-galactosidase from Kluyveromyces lactis (β-gal) on to two different porous carriers, CPC-silica and agarose, is reported. CPC-silica was silanizated and activated with glutaraldehyde. The activation of agarose via a cyanylating agent (CDAP) was optimized. Gel-bound protein and gel-bound activity were both measured directly, allowing the determination of apparent specific activities (S.A.). Higher amounts of β-gal were immobilized on the activated CPC-silica (maximum capacity, 23 mg ml⁻¹ of packed support) than on the CDAP-activated agarose. For the lower enzyme loading assayed (12.6 mg ml⁻¹ packed support), 100% of the enzyme was immobilized but only 34% of its activity was expressed. This inactivation during immobilization was confirmed by the S.A. values (22–29 EU mg⁻¹ for the CPC-derivatives and 80 EU mg⁻¹ for soluble β-gal). The K_app (3.4 mM) for the CDAP-derivative with ONPG as substrate was higher than the K_M value for soluble β-gal (2 mM). When the enzyme loading was increased five-fold, the K_app increased four-fold, to 13 mM. The V_app values for the CPC-derivatives were remarkably lower than the V_max for soluble β-galactosidase. CDAP-derivatives showed better thermal stabilities than CPC-derivatives but neither of them enhanced the stability of the soluble enzyme. When stored at 4°C, the activity of both derivatives remained stable for at least 2 months. Both derivatives displayed high percentages of lactose conversion (90%) in packed bed mini-reactors. Glucose production was 3.3-fold higher for the CPC-derivative than for the CDAP-derivative, as a consequence of the higher flow rates achieved.     

The preparation and properties of the membranal DPNH dehydrogenase from Escherichia coli

1. The membrane bound DPNH oxidase of Escherichia coli can reduce the artificial electron acceptors: ferricyanide, dichlorophenolindophenol (DCIP) and menadione. All three are reduced by the flavoprotein of DPNH oxidase, but at different sites of the enzyme.

2. Freeze-drying of the bacterial membranes causes a selective detachment of DPNH dehydrogenase (DPNH: (acceptor) oxidoreductase, EC 1.6.99.3) from the membranes. This solubilization is accompanied by a decrease of K_m(K₃Fe(CN)₆) from 2.0 to 0.25 mM, while no change is detected in K_m(DPNH). This enzyme is not the DPNH diaphorase found in the bacteria.

3. DPNH dehydrogenase of E. coli is a metalloflavoprotein, containing non-heme iron, labile sulfide, FMN and FAD.

4. Reduction of the enzyme with DPNH in the absence of electron acceptor (ferricyanide or DCIP) causes a rapid and irreversible change to a less active state, Form II. Form II is characterized by a higher K_m(DPNH) and slower v_max., while the K_m(K₃Fe(CN)₆) remains unchanged.

5. The transformation of the enzyme to Form II is accompanied by the reduction of the non-heme iron component. The role of non-heme iron in the enzymic reaction is discussed.     

Partial purification and characterization of fructokinase from developing taproots of sugar beet (Beta vulgaris)

Fructokinase (FK) has been purified from developing sugar beet (Beta vulgaris L.) taproots by ion exchange chromatography and gel filtration. One major isoform was identified. The protein appears to be a dimer (M_r 38 000). Kinetically, the purified sugar beet fructokinase has a pH optimum of 8.5 and a high specificity for fructose (K_m = 0.068 mM). The enzyme can utilise a range of nucleotide triphosphates, although ATP is the most effective. Sugar beet fructokinase is inhibited by fructose concentrations in excess of 0.6 mM. Fructose-6-phosphate and Mg ADP are also inhibitory, but at relatively high concentrations. K⁺ at 10 mM stimulates activity by 30%. Fructokinase activity and the level of FK protein remain high throughout taproot development. Tissue-blots showed that high levels of FK protein were associated with conducting tissues.     

Tachykinin NK-1 receptor probed with constrained analogues of substance P

The action of rotameric probes introduced either in position 7 or 8 in the sequence of substance P (SP) was investigated, i.e. -tetrahydroisoquinoleic acid (Tic), -fluorenylglycine (Flg), -diphenylalanine (Dip), the diastereoisomers of -1-indanylglycine (Ing) and -benz[ƒ]indanylglycine (Bfi), the Z- and E-isomers of dehydrophenylalanine and dehydronaphthylalanine (Δ^ZPhe, Δ^EPhe, Δ^ZNal, Δ^ENal) and (Dmp). The aim of this study was the topographical characterization of the binding subsites of human NK-1 receptor expressed in CHO cells, especially the S₇ and S₈ subsites, corresponding to residues Phe⁷ and Phe⁸ of substance P. According to the binding potencies of these substituted-SP analogues, the S₇ binding subsite is smaller than the S₈ subsite: the S₇ subsite accepts only one aromatic nucleus, while the S₈ can accommodate three coplanar nuclei altogether. These findings are compatible with the idea that the S₈ binding subsite may reside in the extracellular loops of the hNK-1 receptor. NK-1 agonists bind to human NK-1 receptor and activate the production of both inositol phosphates and cyclic AMP. As already quoted for septide, [pGlu⁶, Pro⁹]SP(6–11), discrepancies are observed between affinity (K_i) and activity (EC₅₀) values for IPs production. While a weak correlation between K_i and EC₅₀ values for IPs production could be found (r = 0.70), an excellent correlation could be demonstrated between their affinities (K_i) and their potencies (EC₅₀) for cAMP production (r = 0.97). The high potency (EC₅₀) observed for ‘septide-like’ molecules on PI hydrolysis, compared to their affinity is not an artefact related to the high level of NK-1 receptors expressed on CHO cells since a good correlation was found between EC₅₀ values obtained for PI hydrolysis and those measured for spasmogenic activity in guinea pig ileum bioassay (r = 0.94).

According to the binding potencies of constrained analogues of phenylalanine, the S₇ binding subsite of human NK-1 receptor is small, whereas the S₈, which can accommodate three coplanar nuclei, might probably reside in the extracellular loop. The discrepancies observed between affinity (K_i) and activity (EC₅₀) values for IPs production are not an artefact of CHO cells since a good correlation was found between EC₅₀ for PI hydrolysis and those measured in guinea pig ileum bioassay.     

Induction of glyoxylate cycle enzymes in rat liver upon food starvation

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of foodstarved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The K_m(isocitrate) was 68 μM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a K_m(acetyl-CoA) of 0.2 μM, a K_m(glyoxylate) of 3 mM and a pH optimum of 7.6.     

Methionine analogues as inhibitors of methionyl-tRNA synthetase

A series of methionine analogues have been synthesized as inhibitors of methionyl-tRNA synthetase and evaluated for their inhibitory activities of E. coli methionyl-tRNA synthetase and bacterial growth. Among them, -methionine hydroxamate 20 has proved to be the best inhibitor of the enzyme with K_i = 19 μM and showed a growth inhibition against E.coli JM 109, P. vulganis 6059 and C. freundii 8090.     

Inhibitors of bacterial tyrosyl tRNA synthetase: synthesis of four stereoisomeric analogues of the natural product SB-219383

Synthetic analogues of the microbial metabolite SB-219383 have been synthesised with defined stereochemistry. Densely functionalised hydroxylamine containing amino acids were prepared by the addition of a glycine anion equivalent to sugar-derived cyclic nitrones. One of four stereoisomeric dipeptides incorporating these novel amino acids was found to be a potent and selective inhibitor of bacterial tyrosyl tRNA synthetase, suggesting analogous stereochemistry of the natural product.     

Bioconversion of phospholipids by immobilized phospholipase A2

Phospholipase A₂ selectively hydrolyses the ester linkage at the sn-2 position of phospholipids forming lysocompounds. This bioconversion has importance in biotechnology since lysophospholipids are strong bioemulsifiers. The aim of the present work was to study the kinetic behaviour and properties of immobilized phospholipase A₂ from bee venom adsorbed into an ion exchange support. The enzyme had high affinity for CM-Sephadex^® support and the non-covalent interaction was optimum at pH 8. The activity of immobilized phospholipase A₂ was comparatively evaluated with the soluble enzyme using a phospholipid/Triton X-100 mixed micelle as assay system. The immobilized enzyme showed high retention activity and excellent stability under storage. The activity of the immobilized system remained almost constant after several cycles of hydrolysis. Immobilized phospholipase A₂ was less sensitive to pH changes compared to soluble form. The kinetic parameters obtained (V_max 883.4 μmol mg⁻¹ min⁻¹ and a K_m 12.9 mM for soluble form and V_max = 306 μmol mg⁻¹ min⁻¹ and a K_m = 3.9 for immobilized phospholipase A₂) were in agreement with the immobilization effect. The results obtained with CM-Sephadex^®-phospholipase A₂ system give a good framework for the development of a continuous phospholipid bioconversion process.     

Purification, characterization, and gene cloning of a novel fluoroacetate dehalogenase from Burkholderia sp. FA1

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of the highly stable carbon–fluorine bond in an aliphatic compound. The bacterial isolate FA1, which was identified as Burkholderia, grew on fluoroacetate as the sole carbon source to produce fluoroacetate dehalogenase (FAc-DEX FA1). The enzyme was purified to homogeneity and characterized. The molecular weights were estimated to be 79,000 and 34,000 by gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that the enzyme is a dimer. The purified enzyme was specific to haloacetates, and fluoroacetate was the best substrate. The activities toward chloroacetate and bromoacetate were less than 5% of the activity toward fluoroacetate. The K_m and V_max values for the hydrolysis of fluoroacetate were 5.1 mM and 11 μmol per minute milligram, respectively. The gene coding for the enzyme was isolated, and the nucleotide sequence was determined. The open reading frame consisted of 912 nucleotides, corresponding to 304 amino acid residues. Although FAc-DEX FA1 showed high sequence similarity to fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX H1) (61% identity), the substrate specificity of FAc-DEX FA1 was significantly different from that of FAc-DEX H1: FAc-DEX FA1 was more specific to fluoroacetate than FAc-DEX H1.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.     

Enzyme inhibition by dipeptides containing 2,3-methanophenylalanine, a sterically constrained amino acid

Both isomers of (E)-2,3-methanophenylalanine (^EPhe), a sterically restricted amino acid, were incorporated into peptides in order to examine their possible enzyme inhibitory activity. Both (2R,3S)- and (2S,3R)-^EPhe-Phe(or Leu)-OMe were found to inhibit effectively the hydrolysis of Ac-Tyr-OEt by chymotrypsin in a competitive manner. The ester groups of these dipeptides were quite resistant to chymotrypsin hydrolysis, and the ^EPhe-Phe peptide bond was also entirely stable. The inhibition constant (K_i) of the most potent dipeptide of H-(2R,3S)-^EPhe-Phe-OMe was 0.16 mM at 25°C. The inhibitory action of Phe-containing peptides was found to depend on the configuration of the Phe residue. The electrophilic nature of the cyclopropane ring which is conjugated with both the phenyl ring and the ester carbonyl group appears to be relevant to the inhibitory activity. Fully irreversible inactivation of chymotrypsin was achieved by its incubation with H-(2R,3S)-^EPhe-Leu-OMe. An enzyme carboxylate group is thought to be responsible for nucleophilic attack on the cyclopropane ring leading to irreversible inactivation.