Inhibition of human arginase I by substrate and product analogues

Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to generate l-ornithine and urea. We demonstrate that N-hydroxy-l-arginine (NOHA) binds to this enzyme with K_d = 3.6 μM, and nor-N-hydroxy-l-arginine (nor-NOHA) binds with K_d = 517 nM (surface plasmon resonance) or K_d ≈ 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 Å resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with l-lysine (K_d = 13 μM) is reported at 1.90 Å resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor α-carboxylate and α-amino groups as key specificity determinants of amino acid recognition in the arginase active site.     

Unusual hepatic mitochondrial arginase in an Indian air-breathing teleost,Heteropneustes fossilis: Purification and characterization

A functional urea cycle with both cytosolic (ARG I) and mitochondrial (ARG II) arginase activity is present in the liver of an ureogenic air-breathing teleost, Heteropneustes fossilis. Antibodies against mammalian ARG II showed no cross-reactivity with the H. fossilis ARG II. ARG II was purified to homogeneity from H. fossilis liver. Purified ARG II showed a native molecular mass of 96 kDa. SDS–PAGE showed a major band at 48 kDa. The native enzyme, therefore, appears to be a homodimer. The pI value of the enzyme was 7.5. The purified enzyme showed maximum activity at pH 10.5 and 55 °C. The K_m of purified ARG II for l-arginine was 5.25 ± 1.12 mM. l-Ornithine and N^ω-hydroxy-l-arginine showed mixed inhibition with K_i values 2.16 ± 0.08 and 0.02 ± 0.004 mM respectively. Mn^+ 2 and Co^+ 2 were effective activators of arginase activity. Antibody raised against purified H. fossilis ARG II did not cross-react with fish ARG I, and mammalian ARG I and ARG II. Western blot with the antibodies against purified H. fossilis hepatic ARG II showed cross reactivity with a 96 kDa band on native PAGE and a 48 kDa band on SDS–PAGE. The molecular, immunological and kinetic properties suggest uniqueness of the hepatic mitochondrial ARG II in H. fossilis.     

New pyrazolopyrimidine derivatives as Leishmania amazonensis arginase inhibitors

Arginase performs the first enzymatic step in polyamine biosynthesis in Leishmania and represents a promising target for drug development. Polyamines in Leishmania are involved in trypanothione synthesis, which neutralize the oxidative burst of reactive oxygen species (ROS) and nitric oxide (NO) that are produced by host macrophages to kill the parasite. In an attempt to synthesize arginase inhibitors, six 1-phenyl-1H-pyrazolo[3,4-d]pyrimidine derivatives with different substituents at the 4-position of the phenyl group were synthesized. All compounds were initially tested at 100 µM concentration against Leishmania amazonensis ARG (LaARG), showing inhibitory activity ranging from 36 to 74%. Two compounds, 1 (R=H) and 6 (R=CF₃), showed arginase inhibition >70% and IC₅₀ values of 12 µM and 47 µM, respectively. Thus, the kinetics of LaARG inhibition were analyzed for compounds 1 and 6 and revealed that these compounds inhibit the enzyme by an uncompetitive mechanism, showing K_is values, and dissociation constants for ternary complex enzyme-substrate-inhibitor, of 8.5 ± 0.9 µM and 29 ± 5 µM, respectively. Additionally, the molecular docking studies proposed that these two uncompetitive inhibitors interact with different LaARG binding sites, where compound 1 forms more H-bond interactions with the enzyme than compound 6. These compounds showed low activity against L. amazonensis free amastigotes obtained from mice lesions when assayed with as much as 30 µM. The maximum growth inhibition reached was between 20 and 30% after 48 h of incubation. These results suggest that this system can be promising for the design of potential antileishmanial compounds.     

UDP-galactopyranose mutases from Leishmania species that cause visceral and cutaneous leishmaniasis

Leishmaniasis is a vector-borne, neglected tropical disease caused by parasites from the genus Leishmania. Galactofuranose (Galf) is found on the cell surface of Leishmania parasites and is important for virulence. The flavoenzyme that catalyzes the isomerization of UDP-galactopyranose to UDP-Galf, UDP-galactopyranose mutase (UGM), is a validated drug target in protozoan parasites. UGMs from L. mexicana and L. infantum were recombinantly expressed, purified, and characterized. The isolated enzymes contained tightly bound flavin cofactor and were active only in the reduced form. NADPH is the preferred redox partner for both enzymes. A k_cat value of 6 ± 0.4 s⁻¹ and a K_m value of 252 ± 42 μM were determined for L. infantum UGM. For L. mexicana UGM, these values were ∼4-times lower. Binding of UDP-Galp is enhanced 10–20 fold in the reduced form of the enzymes. Changes in the spectra of the reduced flavin upon interaction with the substrate are consistent with formation of a flavin-iminium ion intermediate.     

Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K_D (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the K_D of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC₅₀ of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k_cat, it decreased the K_m of nelfinavir, suggesting a decrease in catalytic efficiency (k_cat/K_m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.     

Comparative effects of cationic triarylmethane, phenoxazine and phenothiazine dyes on horse serum butyrylcholinesterase

The kinetic effects of a selection of triarylmethane, phenoxazine and phenothiazine dyes (pararosaniline (PR), malachite green (MG), methyl green (MeG); meldola blue (MB), nile blue (NB), nile red (NR); methylene blue (MethB)) and of ethopropazine on horse serum butyrylcholinesterase were studied spectrophotometrically at 25 ^°C in 50 mM MOPS buffer, pH 8, using butyrylthiocholine as substrate. PR, MeG, MB and ethopropazine acted as linear mixed type inhibitors of the enzyme, with respective K_i values of 4.5 ± 0.50 μM, 0.41 ± 0.007 μM, 0.44 ± 0.086 μM and 0.050 ± 0.0074 μM. MG, NB, MethB and NR caused complex, nonlinear inhibition pointing to cooperative binding at two sites. Intrinsic K′ values (≡[I]²_0.5 extrapolated to [S]=0) for MG, NB, NR and MethB were 0.20 ± 0.096 μM, 0.0018 ± 0.0015 μM, 0.92 ± 0.23 μM and 0.23 ± 0.08 μM. NB stood out as a potent inhibitor effective at nM levels. Comparison of inhibitory effects on horse and human serum butyrylcholinesterases suggested that the two enzymes must have distinct microstructural features.     

Human mevalonate diphosphate decarboxylase: characterization, investigation of the mevalonate diphosphate binding site, and crystal structure

Expression in Escherichia coli of his-tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation of the mevalonate diphosphate binding site, and crystal structure determination (2.4 Å resolution). hMDD exhibits V_max = 6.1 ± 0.5 U/mg; K_m for ATP is 0.69 ± 0.07 mM and K_m for (R,S) mevalonate diphosphate is 28.9 ± 3.3 μM. Conserved polar residues predicted to be in the hMDD active site were mutated to test functional importance. R161Q exhibits a ∼1000-fold diminution in specific activity, while binding the fluorescent substrate analog, TNP-ATP, comparably to wild-type enzyme. Diphosphoglycolyl proline (K_i = 2.3 ± 0.3 uM) and 6-fluoromevalonate 5-diphosphate (K_i = 62 ± 5 nM) are competitive inhibitors with respect to mevalonate diphosphate. N17A exhibits a V_max = 0.25 ± 0.02 U/mg and a 15-fold inflation in K_m for mevalonate diphosphate. N17A’s K_i values for diphosphoglycolyl proline and fluoromevalonate diphosphate are inflated (>70-fold and 40-fold, respectively) in comparison with wild-type enzyme. hMDD structure indicates the proximity (2.8 Å) between R161 and N17, which are located in an interior pocket of the active site cleft. The data suggest the functional importance of R161 and N17 in the binding and orientation of mevalonate diphosphate.     

Conformational stability of α-amylase from malted sorghum (Sorghum bicolor): Reversible unfolding by denaturants

α-Amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50 mM Hepes containing 13.6 mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 °C is characterized by two state transition with ΔG (H₂O) of 16.5 kJ mol⁻¹ and 22 kJ mol⁻¹, respectively, at pH 4.8 and pH 7.0 for GuHCl and ΔG (H₂O) of 25.2 kJ mol⁻¹ at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (ΔC_p), the unfolding enthalpy (H_g) and the temperature at ΔG = 0 (T_g) are 17.9 ± 0.7 kJ mol⁻¹ K⁻¹, 501.2 ± 18.2 kJ mol⁻¹ and 337.3 ± 6.9 K at pH 4.8 and 14.3 ± 0.5 kJ mol⁻¹ K⁻¹, 509.3 ± 21.7 kJ mol⁻¹ and 345.4 ± 4.8 K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the ‘B’ domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by ΔG (H₂O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.     

Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg^2 + dependent and catalyzes a similar S_N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give K_chorismate = 20 ± 4 μM, K_Mg^2 + = 294 ± 22 μM, K_L-gln = 11 ± 1 mM, and k_cat = 2.2 ± 0.2 s^− 1 for a random kinetic mechanism. PhzE can use NH₄⁺ as an alternative nucleophile, while Co^2 + and Mn^2 + are alternative divalent metals.     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

Enzyme kinetics of ginsenosidase type IV hydrolyzing 6-O-multi-glycosides of protopanaxatriol type ginsenosides

In this work, the kinetics of ginsenosidase type IV hydrolyzing the 6-O-multi-glycosides of protopanaxatriol type ginsenosides (PPT) from Aspergillus sp.39g strain were investigated. The enzyme molecular weight was about 56 kDa. The enzyme hydrolyzes the 6-O-α-l-(1 → 2)-rhamnoside of ginsenoside Re and 6-O-β-d-(1 → 2)-xyloside of R1 into Rg1, and subsequently hydrolyzes 6-O-β-d-glucoside of Rg1 into F1. The enzyme hydrolyzes 6-O-α-l-(1 → 2)-rhamnoside of Rg2 and 6-O-β-d-(1 → 2)-glucoside of Rf into Rh1, and subsequently hydrolyzes 6-O-β-d-glucoside of Rh1 into its aglycone. The enzyme K_m and V_max for Re were 22.2 mM, and 7.94 mM/h; the K_m and V_max for R1 were 7.06 mM and 1.61 mM/h; the enzyme transformation velocity (V₀) at 5 mM substrate was 1.46 mM/h for Re, and 0.67 mM/h for R1. Therefore, the enzyme hydrolysis on the Re rhamnoside was faster than that on R1 xyloside. The enzyme V₀ on Rg1 was 0.05 mM/h that indicated the enzyme hardly hydrolyzed the 6-O-β-d-glucoside of Rg1. The enzyme kinetic parameters of Rg2 and Rf were 5.74 and 9.43 mM for K_m; 2.70 and 2.84 mM/h for V_max; 1.26 and 0.98 mM/h for V₀ at 5 mM substrate, respectively. Thus the enzyme hydrolysis on Rg2 rhamnoside was faster than that on the glucoside of Rf.     

ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F1-ATPase are affected by inorganic phosphate

The effect of inorganic phosphate (P_i) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F₁-ATPase from beef heart mitochondria (ndMF₁) has been investigated. It is shown for the first time that P_i decreases the apparent rate constant of uni-site ATP binding by ndMF₁ 3-fold with the K_d of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, P_i also shifts equilibrium between bound ATP and ADP + P_i in the direction of ATP synthesis with the K_d of 0.17 ± 0.03 mM. However, 10 mM P_i does not significantly affect ATP binding during multi-site catalysis.     

Giardia lamblia: biochemical characterization of an ecto-ATPase activity

In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78 ± 0.08 nmol Pi × h⁻¹ × 10⁻⁶ cells). The ATP hydrolysis was stimulated by MgCl₂ in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53 ± 0.07 mM. ATP was the best substrate for this enzyme. The apparent K_m for ATP was 0.21 ± 0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg²⁺-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A₁ (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and 5´ AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl₂. Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology.     

Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Structure based virtual screening of 308 307 chemical compounds was performed using the computation tool Autodock 3.0.5 on a WISDOM Production Environment. The top 1468 ranked compounds with free binding energy ranging from −14.0 to −17.09 kcal mol⁻¹ were selected to check the hydrogen bond interaction with amino acid residues in the active site of 3CL^pro. Fifty-three compounds from 35 main groups were tested in an in vitro assay for inhibition of 3CL^pro expressed by Escherichia coli. Seven of the 53 compounds were selected; their IC₅₀ ranged from 38.57 ± 2.41 to 101.38 ± 3.27 μM. Two strong 3CL^pro inhibitors were further identified as competitive inhibitors of 3CL^pro with K_i values of 9.11 ± 1.6 and 9.93 ± 0.44 μM. Hydrophobic and hydrogen bond interactions of compound with amino acid residues in the active site of 3CL^pro were also identified.     

Chemical evaluation of fatty acid desaturases as drug targets in Trypanosoma cruzi

Four positional isomers of Thiastearate (TS) and Isoxyl (Thiocarlide) were assayed as fatty acid desaturase inhibitors in Trypanosoma cruzi epimastigotes. 9-TS did not exert a significant effect on growth of T. cruzi, nor on the fatty acid profile of the parasite cells. One hundred micromolars of 10-TS totally inhibited growth, with an effective concentration for 50% growth inhibition (EC₅₀) of 3.0 ± 0.2 μM. Growth inhibition was reverted by supplementing the culture media with oleate. The fatty acid profile of treated cells revealed that conversion of stearate to oleate and palmitate to palmitoleate were drastically reduced and, as a consequence, the total level of unsaturated fatty acids decreased from 60% to 32%. Isoxyl, a known inhibitor of stearoyl-CoA Δ9 desaturase in mycobacteria, had similar effects on T. cruzi growth (EC₅₀ 2.0 ± 0.3 μM) and fatty acid content, indicating that Δ9 desaturase was the target of both drugs. 12- and 13-TS were inhibitors of growth with EC₅₀ values of 50 ± 2 and 10 ± 3 μM, respectively, but oleate or linoleate were unable to revert the effect. Both drugs increased the percentage of oleate and palmitate in the cell membrane and drastically reduced the content of linoleate from 38% to 16% and 12%, respectively, which is in agreement with a specific inhibition of oleate Δ12 desaturase. The absence of corresponding enzyme activity in mammalian cells and the significant structural differences between trypanosome and mammalian Δ9 desaturases, together with our results, highlight these enzymes as promising targets for selective chemotherapeutic intervention.     

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca²⁺ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca²⁺-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (∼780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca²⁺]_i measurement in GFP expressing cells. The indicators bind Ca²⁺ with either high (K_d = 0.49 ± 0.07 μM; ACR-1) or low affinity (K_d = 6.65 ± 0.13 μM; ACR-1-LA). Chelating Zn²⁺ (K_d = 0.38 ± 0.02 nM) or Mg²⁺ (K_d ∼ 5 mM) slightly raises and binding Co²⁺ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pK_a = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.     

A continuous assay for α-methylacyl-coenzyme A racemase using circular dichroism

α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K_m = 48 ± 5 μM, k_cat = 291 ± 30 s⁻¹), but turned over (2S)-ibuprofenoyl-CoA (K_m = 86 ± 6 μM, k_cat = 450 ± 14 s⁻¹) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His₆-tag had a catalytic efficiency (k_cat/K_m) that was reduced 22% and 47% in the 2S → 2R and 2R → 2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.     

Trypanosoma evansi: Pharmacological evidence of a nicotinic acetylcholine receptor

The role of calcium and its relevance have been deeply revised with respect to trypanosomatids, as the mechanism by which calcium enters trypanosomes was, until now, not well understood. There is evidence supporting the presence of a nAChR in another member of the trypanosomatidae family, Trypanosoma cruzi, these receptors being one entry path to calcium ions. The aims of this work were to determine if there was a nicotinic acetylcholine receptor (nAChR) in Trypanosoma evansi, and to subsequently perform a partial pharmacological characterization of this receptor.After being loaded with FURA-2AM, individual cells of T. evansi, were exposed to cholinergic compounds, and the cells displayed a dose-dependent response to carbachol. This observation indicated that a cholinergic receptor may be present in T. evansi. Although a dose-dependent response to muscarine could not be demonstrated, nicotine could promote an incremental dose-dependent response. The relative potency of this specific agonist of nAChR is in agreement with previous reports. The estimated affinity values were a K_d1 value of 29.6 ± 5.72 nM and a K_d2 value of 315.9 ± 26.6 nM, which is similar to the K_d value reported for the α4 nicotinic receptor. The Hill coefficients were determined to be an n₁ of 1.2 ± 0.3 and an n₂ of 4.2 ± 1.3. Finally, our calculations indicated that there are about 1020 receptors in each T. evansi parasite, which is approximately 15-fold lower than the number reported in Torpedo californica electric cells. These results suggest the presence of a nAChR in T. evansi, which is able to bind nicotinic ligands and induce calcium signals.     

Characterization of recombinant UDP-galactopyranose mutase from Aspergillus fumigatus

UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose, which is an important cell wall component in Aspergillus fumigatus and other pathogenic microbes. A. fumigatus UGM (AfUGM) was expressed in Escherichia coli and purified to homogeneity. The enzyme was shown to function as a homotetramer by size-exclusion chromatography and to contain ∼50% of the flavin in the active reduced form. A k_cat value of 72 ± 4 s⁻¹ and a K_M value of 110 ± 15 μM were determined with UDP-galactofuranose as substrate. In the oxidized state, AfUGM does not bind UDP-galactopyranose, while UDP and UDP-glucose bind with K_d values of 33 ± 9 μM and 90 ± 30 μM, respectively. Functional and structural differences between the bacterial and eukaryotic UGMs are discussed.     

Triose phosphate isomerase from the blood fluke Schistosoma mansoni: Biochemical characterisation of a potential drug and vaccine target

The glycolytic enzyme triose phosphate isomerase from Schistosoma mansoni is a potential target for drugs and vaccines. Molecular modelling of the enzyme predicted that a Ser-Ala-Asp motif which is believed to be a helminth-specific epitope is exposed. The enzyme is dimeric (as judged by gel filtration and cross-linking), resistant to proteolysis and highly stable to thermal denaturation (melting temperature of 82.0 °C). The steady-state kinetic parameters are high (K_m for dihydroxyacetone phosphate is 0.51 mM; K_m for glyceraldehyde 3-phosphate is 1.1 mM; k_cat for dihydroxyacetone phosphate is 7800 s⁻¹ and k_cat for glyceraldehyde 3-phosphate is 6.9 s⁻¹).