Some properties of γ-tocopherol methyltransferase solubilized from spinach chloroplasts

γ-Tocopherol methyltransferase occurs in the chloroplast fraction of spinach leaves. Its specific activity with γ-tocopherol and S-adenosyl-l-methionine was 3.91 nmol hr⁻¹ mg⁻¹ protein. The enzyme was effectively solubilized by 6 mM sodium deoxycholate from the membrane fraction of chloroplasts. The activity was maximum at pH 7.5 and 35°. γ-Tocopherol was preferred to β-tocopherol (25:7). The K_m value for S-adenosyl-l-methionine as methyl donor was 9.1 μM.     

Molecular cloning and characterisation of a novel xanthine oxidase from Cellulosimicrobium cellulans ATCC21606

Xanthine oxidase (XOD) catalyses the oxidation of hypoxanthine into xanthine and xanthine into uric acid. The enzyme plays a key role in the purine metabolic pathway. Despite the presence of different XODs in prokaryotes, the functional and structural knowledge of prokaryotic XODs remain limited (compared with their well-known eukaryotic counterparts), thereby hindering their biochemical analysis and industrial application. Using genetic and biochemical analyses, we identified and characterised recombinant XOD (CcXOD_AB) from Cellulosimicrobium cellulans ATCC21606. Bioinformatics analysis suggests that CcXOD_AB shares low amino acid sequence identities with other XODs. The purified enzyme exhibits the maximum activity at 55 °C and pH 8.0. In addition, CcXOD_AB exhibits moderate thermostability and retains 80.65 % of the original activity after 30 min of incubation at 60 °C. Ca^{2 +} has a slight inhibitory effect, whereas Co^{2 +} and Mn^{2 +} have a strong inhibitory effect on XOD_AB activity. In particular, low Ba²⁺ and Mg^{2 +} concentrations have no effect, whereas high Mg^{2 +} (≥10 mM) and Ba²⁺ (≥2 mM) concentrations show an inhibitory effect on enzyme activity. The K_m and V_max values for xanthine are 131.29 ± 11.09 μmol•L⁻¹ and 15.23 ± 0.65 μmol•L^-1 min⁻¹, respectively. Results indicate that CcXOD_AB is a novel enzyme with potential industrial application.     

Subunit-subunit interactions in TF1 as revealed by ligand binding to isolated and integrated α and β subunits

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF₁ and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (K_D(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The K_D(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The K_D(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF₁ binds 2 mol 1-naphthoyl-ATP per mol enzyme with K_D = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 10⁴ M⁻¹s⁻¹. (6) The rate constant for 1-naphthoyl-ATP binding to TF₁ is 6.6 · 10³ M⁻¹ · s⁻¹ (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (k^A₋₁ = 3 · 10⁻³s⁻¹) and a slower second phase (k^A₋₂ < 0.2 · 10⁻³s⁻¹). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF₁ are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.     

Purification and characterization of laccase secreted by Phellinus linteus MTCC-1175 and its role in the selective oxidation of aromatic methyl group

A laccase from the culture filtrate of Phellinus linteus MTCC-1175 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on DEAE-cellulose. The SDS-PAGE and native-PAGE gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 70 kDa. Using 2.6-dimethoxyphenol, 2.2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine as the substrates, the K _m, k _cat and k _cat/K _m values of the laccase were found to be 160 μM, 6.85 s^?1, 4.28 × 10⁴ M^?1 s^?1, 42 μM, 6.85 s^?1, 16.3 × 10⁴ M^?1 s^?1 and 92 μM, 6.85 s^?1, 7.44 × 10⁴ M^?1 s^?1, respectively. The pH and the temperature optima of the P. linteus MTCC-1175 laccase were 5.0 and 45°C, respectively. The activation energy for thermal denaturation of the enzyme was 38.20 kJ/mole/K. The enzyme was the most stable at pH 5.0 after 1 h reaction. In the presence of ABTS as the mediator, the enzyme transformed toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde and 4-chlorobenzaldehyde, respectively.     

High throughput covalent immobilization process for improvement of shelf-life,operational cycles,relative activity in organic media and enzymatic kinetics of urease and its application for urea removal from water samples

High throughput covalent urease immobilization was performed through the amide bond formation between the urease and the amino-functional MNPs. The enzyme’s performances, including shelf-life, reusability, enzymatic kinetics, and the enzyme relative activity in organic media was improved. At optimal conditions, the immobilization efficiency was calculated about 95.0% with keeping 94.7% of the urease initial specific activity. The optimal pH for maximum activity of the free and immobilized urease was calculated as 7.0 at 37.0 °C and 8.0 at 60.0 °C, respectively. The kinetics studies showed the K_m of 26.0 mM and 8.0 mM and the V_max of 5.31 μmol mg⁻¹ min⁻¹ and 3.93 μmol mg⁻¹ min⁻¹ for the free and immobilized urease, respectively. The ratio K_cat/K_m as a measure of catalytic efficiency and enzyme specificity was calculated as 0.09 mg mL⁻¹ min⁻¹ and 0.22 mg mL⁻¹ min⁻¹ for the free and immobilized urease, respectively, indicating an improvement in the enzymatic kinetics. The shelf-life and operational studies of immobilized urease indicated that approximately 97.7% and 88.5% of its initial activity was retained after 40 days and 17 operational cycles, respectively. The immobilized urease was utilized to urea removal from water samples with an efficiency between 91.5–95.0%.     

Control of denitrification enzyme activity in a streamside soil

Progress curve analysis of NO₃⁻ and NO₂⁻ reduction in surface soil samples from a streamside soil gave K_m values of 4.24 and 6.33 μM, and V_max values of 2.16 and 1.83 μmol l⁻¹ min⁻¹, respectively. Recoveries of reduced NO₃⁻ and NO₂⁻ as gaseous N averaged 82 and 108%. The unrecovered NO₃⁻-N was presumably dissimilated to NH₄⁺-N. The idenitification enzyme activity (DEA) was examined throughout a year and showed seasonal and spatial variabilities of only 10% to 26%, suggesting a high persistency of denitrifying enzymes. Soil moisture and DEA correlated significantly (r = 0.7671; P<0.01). The DEA in saturated subsoil also showed a relatively little variation, with spatial variabilities of between 28 and 38%. Amendment with NO₃⁻ rarely enhanced the acctivity more than two-fold at either depth. Addition of glucose increased the activity 2.3 and 2.5 times in the surface soil and suboil respectively, indicating a moderate carbon limitation of denitrification. The activation energy of DEA was found to be 64.9 kJ mol⁻¹ and Q₁₀ values for the 2–12°C and 12–22°C temperature ranges were 2.71 and 2.53, respectively. Extrapolation suggested there would be a 4.4-fold increase in DEA if the temperature was changed from 0 to 15°C. Substrate diffusion limited the denitrification 10 to 25 fold.Thus, under anaerobic moist conditions it appears that changes in denitrification might primarily be due to varying diffusion of substrates into the anaerobic soil centers. Over a year, fluctuations in DEA, temperature changes and fluctuations of electron-acceptor and -donor supply will only have a minor effect on natural denitrification activity.     

Characterization and directed evolution of BliGO,a novel glycine oxidase from Bacillus licheniformis

Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40 °C. Interestingly, BliGO retained 60% of the maximum activity at 0 °C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (K_m, k_cat and k_cat/K_m) of BliGO were 11.22 mM, 0.08 s⁻¹, and 0.01 mM⁻¹ s⁻¹, respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM⁻¹ s⁻¹) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency.     

Kinetics and mechanism of the decomposition of μ-amido-μ-hydroxo(tetraamminecobalt(III))(tetraaquacobalt(III)) ion in acidic aqueous solution

The title complex undergoes decomposition in acidic aqueous solution resulting in equimolar concentration of aquapentaamminecobalt(III) and hexa- aquacobalt(II). The kinetic studies over the ranges of 0.048 M ⩽ [H⁺] ⩽ 0.385 M, 25 ⩽ θ_c ⩽ 41.5°C and at I = 0.5 M reveals that the intricate mechanism involves protonation equilibrium of the title complex, followed by a rate determining bridge cleavage. The further follow-up reaction is a fast electron transfer process to form products. The rate expression derived from the mechanism is k_obs = k₁K₁[H⁺]/(1 + K₁[H⁺]) where the values of k, and K, are found to be 8.9 × 10⁻⁴ s⁻¹ and 3.5 M⁻¹ respectively at 25 °C. The results are compared with that obtained for the decomposition reactions of mononuclear aquaammine complexes of cobalt(III).     

Interaction of bilirubin with brain capillaries and its toxicity

The interaction of bilirubin with isolated brain capillaries, and the effect of bilirubin on the uptake of 2-deoxyglucose by the capillaries were investigated with 1-month-old Sprague-Dawley rats. The binding of bilirubin to the brain capillaries was observed only at a molar ratio of bilirubin to bovine serum albumin higher than 1.0. An absorption spectrum with a microspectrophotometer of the bilirubin-capillary complex showed a broad absorption maximum from 425 to 440 nm with a shoulder near 490 nm, but no shoulder was observed in the case of the bilirubin emulsion. The bilirubin binding activity was dependent on pH and temperature of the medium, but was not affected by sulfhydryl blocking agents such as p-chloromercuribenzoate and N-ethylmaleimide. Bilirubin saturation kinetics gave an apparent K_m for bilirubin of 61.7 μM. Release of the bilirubin from the brain capillaries to the medium was observed at 37°C but not at 4°C. The uptake of 2-deoxyglucose by the isolated brain capillaries was inhibited by bilirubin in a noncompetitive manner, giving an apparent K_i for the pigment of 137 μM.These results suggest that bilirubin may be responsible for the decreased 2-deoxyglucose uptake in the brain capillaries by disturbing the membrane structure of the capillary endothelial cells.     

Kinetic constants for the inhibition of camel retinal acetylcholinesterase by the carbamate insecticide lannate

We have designed this study to determine various kinetic parameters of camel retinal membrane‐bound acetylcholinesterase (AChE; EC 3.1.1.7) inhibition by carbamate insecticide lannate [methyl N‐{{(methylamino)carbonyl}oxy} ethanimidothioate]. All these kinetic constants were derived by simple graphical methods. The value of kinetic parameters was estimated as follows: 0.061 (μM)⁻¹, 1.14 (μM)⁻¹, 0.216 μM, 0.016 min⁻¹, 0.0741 (μM min)⁻¹, 0.746 μM, and 4.42 μM for velocity constant (K_v), new inhibition constant (K_nic), dissociation constant (K_d), carbamylation rate constant (k_2c), overall carbamylation rate constant (k′2 ), 50% inhibition constant (K_I50), and 99% inhibition constant (K_I99), respectively. These unique methods may be used to estimate such kinetic parameters for time‐dependent inhibition of enzymes by variety of chemicals, insecticides, herbicides, and drugs. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 41–46, 1999     

Tissue kallikrein processes small proenkephalin peptides

Tissue kallikrein may play a role in processing precursor polypeptide hormones. We investigated whether hydrolysis of natural enkephalin precursors, peptide F and bovine adrenal medulla docosapeptide (BAM-22P), by hog pancreatic kallikrein is consistent with this concept. Incubation of peptide F with this tissue kallikrein resulted in the release of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin. Met⁵-Lys⁶-enkephalin was the main peptide released, indicating that the major cleavage site was between two lysine residues. At 37°C and pH 8.5, the K_M values for formation of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin were 129 and 191 μM, respectively. Corresponding k_cat values were 0.001 and 0.03 s⁻¹ and k_cat/K_M ratios were 8 and 1.6·10² M⁻¹ · s⁻¹, respectively. Cleavage of peptide F at acidic pH (5.5) was negligible. When BAM-22P was used as a substrate, Met⁵-Arg⁶-enkephalin was released, thus indicating cleavage between two arginine residues. At pH 8.5, K_M was 64 μM, k_cat was 4.5 s⁻¹, and the k_cat/K_M ratio was 7 · 10⁴ M⁻¹ · s⁻¹. At 5.5, the pH of the secretory granules, K_M, k_cat and k_cat/K_M were 184 μM, 1.9 s⁻¹ and 10⁴ M⁻¹ · s⁻¹, respectively. It is unlikely that peptide F could be a substrate for kallikrein in vivo; however, tissue kallikrein could aid in processing proenkephalin precursors such as BAM-22P by cleaving Arg-Arg peptide bonds.     

Thermodynamics and coordination characteristics of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 extraction complex

The extraction equilibrium of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 complex was carried out in the crown ether1,2-dichloroethaneHCl aqueous solution system at different temperatures. The extraction complex has the overall composition (L)₂·(H₃O⁺·χH₂O)₂·UO₂Cl₄²⁻ (L = dicyclohexano-24-crown-8). The values of the extraction equilibrium constants (K_ex) increase steadily with a decrease in temperature: 13.5 (298 K), 7.96 (301 K), 4.20 (303 K) and 2.07 (305 K). A plot of log K_ex against 1/T shows a straight line. The value of the enthalpy change, ΔH°, was calculated from the slope and equals −212 kJ mol⁻¹. The value of the entropy change, ΔS°, was calculated from ΔH° and K_ex and equals −690 J K⁻¹ mol⁻¹, whereas ΔG° = −6.45 kJ mol⁻¹. Comparing these thermodynamic parameters with those of the dicyclohexano-18-crown-6 isomer A [1] (ΔS° = −314 J K⁻¹ mol⁻¹, ΔH° = −101 kJ mol⁻¹ and ΔG° = −8.37 kJ mol⁻¹), it can be seen that ΔH° and ΔS° are more negative for the former than for the latter, and both are enthalpy-stabilized complexes. The molecular structure of the complex has the feature that there are two H₅O₂⁺ ions in it, in contrast to the H₃O⁺ ions in the dicyclohexano-18-crown-6 isomer A complex [1]. Each of the H₅O₂⁺ ions is held in the crown ether cavity by four hydrogen bonds. The H₅O₂⁺ ion has a central bond. The uranium atom forms UO₂Cl₄²⁻ as a counterion away from the crown ether. The formation of this complex is in good agreement with more negative entropy change and less negative free energy change, as mentioned above.     

Purification,catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli

3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg²⁺ or Mn²⁺, with Mn²⁺ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40°C the K_m of E. coli IPMDH was 105 μM for IPM and 321 μM for NAD, the k_cat was 69 s⁻¹. The half denaturationn temperature was 64°C, which was 20°C lower than that of the thermophile enzyme.     

Enzymic synthesis of lignin precursors. Purification of properties of the S-adenosyl-l-methionine: caffeic acid 3-O- methyltransferase from soybean cell suspension cultures.

A 3-O-methyltransferase which catalyzes the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified about 60-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The enzyme utilized, in addition to caffeic acid (K_m = 133 μM), 5-hydroxyferulic acid (K_m = 55 μM), 3,4,5-trihydroxy-cinnamic acid (K_m = 100 μM), and protocatechualdehyde (K_m = 50 μM) as substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of ferulic acid, of ortho-, meta-, and para-coumaric acids, and of the flavonoid compounds quercetin and luteolin. The methylation of caffeic acid and 5-hydroxyferulic acid showed a pH optimum at 6.5–7.0. No stimulation of the reaction velocity was observed when Mg²⁺ ions were added. EDTA did not inhibit the reaction. The K_m for S-adencsyl-l-methionine was 15 μm. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine (K_i = 6.9 μM).     

Kinetics of the decomposition of hydrogen peroxide in the presence of ethylenediaminetetraacetatocerium(IV) complex

The rate of reaction of [Ce(EDTA)(OH)_nⁿ⁻] with H₂O₂ in 0.10 M KNO₃ solution was investigated at various temperatures. The presence of a peroxy intermediate is inferred from spectrophotometric measurements. The general rate equation,

is valid for pH 7-9 with n= 1 and 2 complexes involved. The rate constants k_l and k₂ were determined at 25 °C to be 0.054 and 0.171 M⁻¹ s⁻¹ respectively. The corresponding activation enthalpies, as calculated from Arrhenius plots, were δH₁^#= 51.3 ± 14.8 and δH₂^#= 41.8 ± 5.3 kJ m⁻¹ and the activation entropies were δS₁^#=-97 ± 47 and ΔS₂^#=−119±17 J K⁻¹ m⁻¹.     

Purification and characterization of two thermostable laccases with high cold adapted characteristics from Pycnoporus sp. SYBC-L1

The white-rot fungus Pycnoporus sp. SYBC-L1 produced large amount of laccase in submerged fermentation. Two laccase isozymes (LacI and LacII) were purified using (NH₄)₂SO₄ fractionation, DEAE-cellulose and Sephadex G-100 column chromatography. The molecular masses of LacI and Lac II were 55.89 and 63.07 kDa, respectively by SDS-PAGE. Both the laccases showed acidic pH optima and high catalytic activities at low temperature for oxidations of 2,6-dimethoxyphenol (DMP), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonate acid) (ABTS), syringaldazine and guaiacol. LacI and LacII were not only with high cold adaptation, but also fairly stable at high temperature. The half-lives of LacI at 50, 60 and 70 °C were 69.31, 2.58 and 0.13 h, respectively, whereas LacII was more stable with half-lives of 256.72, 21.00 and 2.06 h respectively. The best substrates for the enzymes were both found to be ABTS, in which the K_m values of LacI and LacII were 0.0166 and 0.0435 mM and the catalytic efficiencies were 19640.36 and 31172.64 S⁻¹ mM⁻¹, respectively. EDTA and low concentration of Cu²⁺ and Mn²⁺ almost had non-inhibitions on their activities. LacII with syringaldehyde efficiently decolorized Remazol Brilliant Blue R. The high thermostabilities as well as cold adapted properties made Pycnoporus sp. SYBC-L1 laccases to be excellent candidates in harsh industry.     

Efficient synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose by using new 2-deoxy-d-ribose-5-phosphate aldolase from Streptococcus suis with moderate activity and aldehyde tolerance

The enzyme 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a useful tool for synthesizing statin side-chain intermediates. In this work, we identified the DERA from Streptococcus suis (SsDERA) by structural and sequence alignment and highly expressed it in Escherichia coli BL21. The recombinant SsDERA had a specific activity of 18.2 U mg⁻¹, K_M of 0.8 mM, and V_max of 32.9 μmol min⁻¹ mg⁻¹ toward 2-deoxy-d-ribose-5-phosphate under the optimal conditions: 40 °C and pH 7.0. The enzyme retained 23.3 % activity after incubation in 200 mM acetaldehyde for 2 h and 58.2 % activity in 100 mM chloroacetaldehyde for 2 h. The enzyme showed moderate activity and aldehyde tolerance compared with reported DERAs. The SsDERA-catalyzed reaction between 200 mM acetaldehyde and 100 mM chloroacetaldehyde generated (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose in 76 % yield in 8 h. This work provides a new DERA for the synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose, which is a potential candidate for the industrial synthesis of statin intermediates.     

Immobilisation of Candida rugosa lipase on polyhydroxybutyrate via a combination of adsorption and cross-linking agents to enhance acylglycerol production

In the present study, a combination of immobilisation processes was utilised to prepare robust biocatalysts. First, lipase from Candida rugosa was adsorbed on polyhydroxybutyrate (PHB) particles, followed by cross-linking with glutaraldehyde. Conditions for creating immobilised lipase involved the addition of 0.6 M glutaraldehyde and 45 U mL⁻¹ lipase while mixing at 150 rpm (4 °C) for 30 min. These conditions produced the highest yield of immobilised lipase (92 %) and the highest levels of activity (1.94 mg g⁻¹ support). At 40 °C and pH 9 the immobilised enzyme was optimally active with a K_m and V_maxat 1.2 mM and 2.5 × 10^-3 mmol min⁻¹, respectively. The use of immobilised lipase improved thermal stability, storage stability, and reusability.The immobilised lipase retained 80 % of its activity after incubation at 30–60 °C for 2 h and 4 °C for 30 d in 0.2 M sodium phosphate buffer (pH 7.0). Moreover, the immobilised enzyme retained 50 % of its activity after more than 14 cycles under optimal conditions. The immobilised lipase was used to produce monoacylglycerol MAG. The existence of a carbonyl group at 1,743 and 1,744 cm⁻¹ was identified using attenuated total reflectance (ATR)-Fourier transformed infrared spectroscopy. Results showed that 48 % MAG was produced.     

Kinetics of imidazole addition to the axial site of the iron(II) complex of 2, 3, 9,10-tetraphenyl-1,4, 8,11-tetraaza-1,3, 8,10-cyclotetradecatetraene in dimethyl sulfoxide. Evidence for a dissociative-interchange mechanism

The axial adduct formation of the iron(II) complex of 2,3,9,10-tetraphenyl-l,4,8,11-tetraaza-1,3,8,10-cyclotetradecatetraene (L) with imidazole in dimethyl sulfoxide has been investigated spectrophotometrically at various temperatures and pressures. In the presence of a large excess of imidazole the reaction with the two phases has been observed. The first faster reaction is the formation of the monoimidazole complex of FeL²⁺, and the second slower reaction corresponds to the formation of the bisimidazole complex. Activation parameters are as follows: for the first step with k₁ (25.0°C) = (6.8 ±0.2)×10⁵ mol⁻¹ kg s⁻¹, ΔH³₁ = 47.5 ± 4.9 kJ mol⁻¹, ΔS³₁ = 26±16 J K⁻¹ mol⁻¹, and ΔV³₁ (30.0°C) = 27.2±1.5 cm³ mol⁻¹; for the second step with k₂ (25.0°C) = 26.8±0.8 mol⁻¹ kg s⁻¹, ΔH³₂ = 91.6± 0.8 kJ mol⁻¹, ΔS³₂ = 90±3 J K⁻¹ mol⁻¹, and ΔV³₂ (35.0°C) = 21.8±0.9 cm³ mol⁻¹. The large positive activation volumes strongly indicate a dissociative character of the activation process.     

5′-Haloacetamido-5′-deoxythymidines: novel inhibitors of thymidylate synthase

5′-Bromoacetamido-5′-deoxythymidine (BAT), 5′-iodoacetamido-5′-deoxythymidine (IAT), 5′-chloroacetamido-5′-deoxythymidine (CAT) and [¹⁴C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were invesatigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT > IAT > CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4–8 fold higher than it was in the absence of dUMP. The I₅₀ values for BAT were 1·10⁻⁵ M and 1.2·10⁻⁶ M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thynmidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The K_m for the enzyme was 9.2 μM, and the K_i determined for competitive inhibition by BAT was 5.4 μM. Formation of a tight, irreversible compledx is referred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min⁻¹, and the steady-state constant of inactivation, K_i, was estimated to be 6.6 μM. The 5′-haloacetamido-5′-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.