Showing papers on "Non-competitive inhibition published in 1996"

Midazolam Hydroxylation by Human Liver Microsomes In Vitro: Inhibition by Fluoxetine, Norfluoxetine, and by Azole Antifungal Agents

Abstract: Biotransformation of the imidazobenzodiazepine midazolam to its alpha-hydroxy and 4-hydroxy metabolites was studied in vitro using human liver microsomal preparations. Formation of alpha-hydroxy-midazolam was a high-affinity (Km = 3.3 mumol/L) Michaelis-Menten process coupled with substrate inhibition at high concentrations of midazolam. Formation of 4-hydroxy-midazolam had much lower apparent affinity (57 mumol/L), with minimal evidence of substrate inhibition. Based on comparison of Vmax/Km ratios for the two pathways, alpha-hydroxy-midazolam formation was estimated to account for 95% of net intrinsic clearance. Three azole antifungal agents were inhibitors of midazolam metabolism in vitro, with inhibition being largely consistent with a competitive mechanism. Mean competitive inhibition constants (Ki) versus alpha-hydroxy-midazolam formation were 0.0037 mumol/L for ketoconazole, 0.27 mumol/L for itraconazole, and 1.27 mumol/L for fluconazole. An in vitro-in vivo scaling model predicted inhibition of oral midazolam clearance due to coadministration of ketoconazole or itraconazole; the predicted inhibition was consistent with observed interactions in clinical pharmacokinetic studies. The selective serotonin reuptake inhibitor (SSRI) antidepressant fluoxetine and its principal metabolite, norfluoxetine, also were inhibitors of both pathways of midazolam biotransformation, with norfluoxetine being a much more potent inhibitor than was fluoxetine itself. This finding is consistent with results of other in vitro studies and of clinical studies, indicating that fluoxetine, largely via its metabolite norfluoxetine, may impair clearance of P450-3A substrates.

Inhibition of thyroid peroxidase by dietary flavonoids.

Abstract: Flavonoids are widely distributed in plant-derived foods and possess a variety of biological activities including antithyroid effects in experimental animals and humans. A structure-activity study of 13 commonly consumed flavonoids was conducted to evaluate inhibition of thyroid peroxidase (TPO), the enzyme that catalyzes thyroid hormone biosynthesis. Most flavonoids tested were potent inhibitors of TPO, with IC50 values ranging from 0.6 to 41 microM. Inhibition by the more potent compounds, fisetin, kaempferol, naringenin, and quercetin, which contain a resorcinol moiety, was consistent with mechanism-based inactivation of TPO as previously observed for resorcinol and derivatives. Other flavonoids inhibited TPO by different mechanisms, such as myricetin and naringin, showed noncompetitive inhibition of tyrosine iodination with respect to iodine ion and linear mixed-type inhibition with respect to hydrogen peroxide. In contrast, biochanin A was found to be an alternate substrate for iodination. The major product, 6,8-diiodo-biochanin A, was characterized by electrospray mass spectrometry and 1H-NMR. These inhibitory mechanisms for flavonoids are consistent with the antithyroid effects observed in experimental animals and, further, predict differences in hazards for antithyroid effects in humans consuming dietary flavonoids. In vivo, suicide substrate inhibition, which could be reversed only by de novo protein synthesis, would be long-lasting. However, the effects of reversible binding inhibitors and alternate substrates would be temporary due to attenuation by metabolism and excretion. The central role of hormonal regulation in growth and proliferation of thyroid tissue suggests that chronic consumption of flavonoids, especially suicide substrates, could play a role in the etiology of thyroid cancer.

Role of cytochrome P450 3A4 in human metabolism of MK-639, a potent human immunodeficiency virus protease inhibitor.

Abstract: MK-639 (L-735,524) is a potent human immunodeficiency virus protease inhibitor under investigation in the treatment of acquired immunodeficiency syndrome. Five in vitro approaches have been used to identify the cytochrome P450 isoform(s) responsible for the human microsomal oxidative metabolism of MK-639. These approaches are: 1) chemical inhibition; 2) immunochemical inhibition; 3) metabolism by cDNA-expressed human cytochrome P450 enzymes; 4) a correlation analysis; and 5) competitive inhibition of marker activities. Ketoconazole and troleandomycin, both selective inhibitors for cytochrome P450 3A4 (CYP3A4), markedly inhibited the formation of all oxidative metabolites of MK-639; whereas other inhibitors (furafylline, sulfaphenazole, quinidine, S-mephenytoin, and diethyldithiocarbamate) had little effect on MK-639 metabolism. This suggested the involvement of CYP3A4 in MK-639 metabolism. Consistent with this, an anti-rat CYP3A1 rabbit polyclonal antibody, which shows a cross-reactive inhibition of CYP3A4-dependent testosterone 6beta-hydroxylation in human liver microsomes, completely inhibited MK-639 metabolism. Human recombinant CYP3A4 showed a high metabolic activity to form all MK-639 metabolites found in native human liver microsomes. In addition, the formation of individual MK-639 metabolites correlated well with each other and with testosterone 6beta-hydroxylation in 12 different human liver microsomes, whereas no correlation was observed between MK-639 metabolite formation and bufuralol 1'-hydroxylation (or tolbutamide methyl hydroxylation). Furthermore, MK-639 strongly inhibited testosterone 6beta-hydroxylation in a concentration-dependent manner. Kinetic analysis showed that MK-639 is a very potent competitive inhibitor for testosterone 6beta-hydroxylation, with a Ki value of approximately 0.5 mu M. Collectively, these results consistently indicate that CYP3A4 is the isoform responsible for the oxidative metabolism of MK-639 in human liver microsomes.

Formation of (R)-8-hydroxywarfarin in human liver microsomes. A new metabolic marker for the (S)-mephenytoin hydroxylase, P4502C19.

Abstract: Kinetic studies demonstrate that two forms of human liver cytochrome P450 are responsible for the formation of (R)-8-hydroxywarfarin: a low-affinity enzyme (KM approximately 1.5 mM), previously identified as P4501A2; and a high-affinity enzyme (KM = 330 microM), now identified as P4502C19 on the basis of the following evidence. In crossover inhibition studies with P4501A2-depleted human liver microsomes between (R)-warfarin and (S)-mephenytoin, reciprocal competitive inhibition was observed. Apparent KM values for (S)-mephenytoin-4'-hydroxylation (52-67 microM) were similar to the determined Ki values (58-62 microM) for (S)-mephenytoin inhibition of (R)-8-hydroxywarfarin formation. Similarly, the apparent KM for (R)-warfarin 8-hydroxylation in furafylline-pretreated microsomes (KM = 289-395 microM) was comparable with the Ki values (280-360 microM) for (R)-warfarin inhibition of (S)-4'-hydroxymephenytoin formation. Inhibition studies with tranylcypromine, a known inhibitor of (S)-mephenytoin hydroxylase activity, and either substrate in three different microsomal preparations yielded nearly identical inhibitory constants: Ki = 8.7 +/- 1.6 microM for inhibition of (S)-4'-hydroxymephenytoin formation and 8.8 +/- 2.5 microM for inhibition of (R)-8-hydroxywarfarin formation. In addition, fluconazole, a potent inhibitor of (R)-warfarin 8-hydroxylation, Ki = 2 microM, was found to inhibit (S)-mephenytoin hydroxylation with an identical Ki (2 microM). Finally, a strong correlation between (S)-mephenytoin 4-hydroxylation and (R)-warfarin 8-hydroxylation activities in furafylline-pretreated microsomes was demonstrated in 14 human liver microsomal preparations (r2 = 0.97).

Phosphinate Inhibitors of the D-Glutamic Acid-Adding Enzyme of Peptidoglycan Biosynthesis.

Abstract: We report the synthesis and initial evaluation of the first effective inhibitors of the D-glutamic acid-adding enzyme (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase or MurD). This enzyme plays a key role in bacterial peptidoglycan biosynthesis and is therefore a target for antibiotic design. Phosphinic acid 3 is a dipeptide analog linked to uridine diphosphate by a hydrophobic spacer. It is a good inhibitor of the enzyme (IC(50) = 0.68 mM) as it closely resembles the tetrahedral intermediate that is presumed to form in the ligation reaction. Compound 4 lacks the terminal UMP group, and compound 5 lacks both the linker and UDP functionalities. These are less effective inhibitors of the enzyme with IC(50) values of 29 mM and >1 mM, respectively. Preincubation of the enzyme in the presence of inhibitor 3 and ATP does not result in irreversible inhibition or in the formation of a slowly decomplexing species, suggesting that the phosphinic acid is not phosphorylated in the active site.

A model for the regulation of D-3-phosphoglycerate dehydrogenase, a Vmax-type allosteric enzyme.

Abstract: Escherichia coli D-3-phosphoglycerate dehydrogenase (ePGDH) is a tetramer of identical subunits that is allosterically inhibited by L-serine, the end product of its metabolic pathway. Because serine binding affects the velocity of the reaction and not the binding of substrate or cofactor, the enzyme is classified as of the Vmax type. Inhibition by a variety of amino acids and analogues of L-serine indicate that all three functional groups of serine are required for optimal interaction. Removing or altering any one functional group results in an increase in inhibitory concentration from micromolar to millimolar, and removal or alteration of any two functional groups removes all inhibitory ability. Kinetic studies indicate at least two serine-binding sites, but the crystal structure solved in the presence of bound serine and direct serine-binding studies show that there are a total of four serine-binding sites on the enzyme. However, approximately 85% inhibition is attained when only two sites are occupied. The three-dimensional structure of ePGDH shows that the serine-binding sites reside at the interface between regulatory domains of adjacent subunits. Two serine molecules bind at each of the two regulatory domain interfaces in the enzyme. When all four of the serines are bound, 100% inhibition of activity is seen. However, because the domain contacts are symmetrical, the binding of only one serine at each interface is sufficient to produce approximately 85% inhibition. The tethering of the regulatory domains to each other through multiple hydrogen bonds from serine to each subunit apparently prevents the body of these domains from undergoing the reorientation that must accompany a catalytic cycle. It is suggested that part of the conformational change may involve a hinge formed in the vicinity of the union of two antiparallel beta-sheets in the regulatory domains. The tethering function of serine, in turn, appears to prevent the substrate-binding domain from closing the cleft between it and the nucleotide-binding domain, which may be necessary to form a productive hydrophobic environment for hydride transfer. Thus, the structure provides a plausible model that is consistent with the binding and inhibition data and that suggests that catalysis and inhibition in this rare Vmax-type allosteric enzyme is based on the movement of rigid domains about flexible hinges.

2-Amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo.

Abstract: 1. The ability of 2-amino-4-methylpyridine to inhibit the catalytic activity of the inducible NO synthase (NOS II) enzyme was characterized in vitro and in vivo. 2. In vitro, 2-amino-4-methylpyridine inhibited NOS II activity derived from mouse RAW 264.7 cells with an IC50 of 6 nM. Enzyme kinetic studies indicated that inhibition is competitive with respect to arginine. 2-Amino-4-methylpyridine was less potent on human recombinant NOS II (IC50 = 40 nM) and was still less potent on human recombinant NOS I and NOS III (IC50 = 100 nM). NG-monomethyl-L-arginine (L-NMMA), N6-iminoethyl-L-lysine (L-NIL) and aminoguanidine were much weaker inhibitors of murine NOS II than 2-amino-4-methylpyridine but, unlike 2-amino-4-methylpyridine, retained similar activity on human recombinant NOS II. L-NMMA inhibited all three NOS isoforms with similar potency (IC50S 3-7 microM). In contrast, compared to activity on human recombinant NOS III, L-NIL displayed 10 x selectivity for murine NOS II and 11 x selectivity for human recombinant NOS II while aminoguanidine displayed 7.3 x selectivity for murine NOS II and 3.7 x selectivity for human recombinant NOS II. 3. Mouse RAW 264.7 macrophages produced high levels of nitrite when cultured overnight in the presence of lipopolysaccharide (LPS) and interferon-gamma. Addition of 2-amino-4-methylpyridine at the same time as the LPS and IFN-gamma, dose-dependently reduced the levels of nitrite (IC50 = 1.5 microM) without affecting the induction of NOS II protein. Increasing the extracellular concentration of arginine decreased the potency of 2-amino-4-methylpyridine but at concentrations up to 10 microM, 2-amino-4-methylpyridine did not inhibit the uptake of [3H]-arginine into the cell. Addition of 2-amino-4-methylpyridine after the enzyme was induced also dose-dependently inhibited nitrite production. Together, these data suggest that 2-amino-4-methylpyridine reduces cellular production of NO by competitive inhibition of the catalytic activity of NOS II, in agreement with results obtained from in vitro enzyme kinetic studies. 4. When infused i.v. in conscious unrestrained rats, 2-amino-4-methylpyridine inhibited the rise in plasma nitrate produced in response to intraperitoneal injection of LPS (ID50 = 0.009 mg kg-1 min-1). Larger doses of 2-amino-4-methylpyridine were required to raise mean arterial pressure in untreated conscious rats (ED50 = 0.060 mg kg-1 min-1) indicating 6.9 x selectivity for NOS II over NOS III in vivo. Under the same conditions, L-NMMA was nonselective while L-NIL and aminoguanidine displayed 5.2 x and 8.6 x selectivity respectively. All of these compounds caused significant increases in mean arterial pressure at doses above the ID50 for inhibition of NOS II activity in vivo. 5. 2-Amino-4-methylpyridine also inhibited LPS-induced elevation in plasma nitrate after either subcutaneous (ID50 = 0.3 mg kg-1) or oral (ID50 = 20.8 mg kg-1) administration. 6. These data indicate that 2-amino-4-methylpyridine is a potent inhibitor of NOS II activity in vitro and in vivo with a similar degree of isozyme selectivity to that of L-NIL and aminoguanidine in rodents.

Non‐Structural Protein 3 of Hepatitis C Virus Inhibits Phosphorylation Mediated by cAMP‐Dependent Protein Kinase

Abstract: Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500, located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKA) and to the autophos-phorylation site in the hinge region of the PKA type II regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189–1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-(1189–1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(1189–1525). Binding studies with PKA and the non-hydrolysable ATP analogue [14C]fluorosulfonylbenzoyladenosine and [3H]cAMP do not reveal any influence of the short HCV-derived peptides or HCV-polyprotein-(1189–1525) upon the affinity of PKA for these nucleotides. The complex interactions of the NS3 fragments could influence one of the most important signal pathways of the cell and, therefore, could possibly provide new pathological mechanisms for HCV infections of liver.

Purification and characterization of fructan: Fructan fructosyl transferase from chicory (Cichorium intybus L) roots

Abstract: Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 μmol·(mg protein) −1·min−1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization ≥ 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited β-fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.

Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits

Abstract: A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fold purification and two isoenzymes designated fructokinase I and II (FKI and FKII) were separated by anion-exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose-12 indicated that for both fructokinases the functional form is a dimer. Two dimensional isoelectric focusing/SDS-PAGE combined with immunoblotting showed that FKI has two components with isoelectric points (pIs) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that the components of FKI match the more alkaline components of FKII. The activity of both fructokinases increased with increasing pH to around 8.0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with Km values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants (Ki) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and Km values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05–0.2 mM), fructose exhibited noncompetitive inhibition with respect to ATP for both fructokinases. This inhibition pattern changed to uncompetitive when higher fructose concentrations (0.5–10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding first. Inhibition by ADP was also affected by the fructose concentrations. At 0.5 mM fructose, FKI showed non-competitive inhibition by ADP with respect to ATP and this inhibition changed to uncompetitive when 3 mM fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both fructokinases suggest that this enzyme might have a relevant role in carbon metabolism during tomato fruit development.

Kinetics and mechanisms of activation and inhibition of porcine liver fructose-1,6-bisphosphatase by monovalent cations.

Abstract: K+ and Li+ were used to study the kinetic effects of monovalent cations on porcine liver fructose-1,6-bisphosphatase (FBPase). At saturating fructose 1,6-bisphosphate (FBP) concentrations, Li+ was found to be a linear noncompetitive inhibitor with respect to Mg2+. K+ was found to activate the wild-type enzyme at low concentrations (K(m) = 17 mM) and to inhibit the enzyme at high concentrations (K(IK+) = 68mM). A steady-state random ter mechanism was proposed, and a mathematical equation was derived to account for the Mg2+ and K+ kinetics and activation of FBPase. Interestingly, when Glu280 was mutated to glutamine by site-directed mutagenesis, K+ lost the ability to activate the enzyme and became a noncompetitive inhibitor with respect to Mg2+. These kinetic data suggest that K+ has two distinct sites. One is a high-affinity activation site and the other a low-affinity inhibition site. Glu280 is essential for allowing K+ to bind at the activation site. Due to the geometric constraints and its small atomic radius, Li+ can bind only at the inhibitory site. It is postulated that monovalent cations activate FBPase by helping the Arg276 residue "deshield" the partial negative charge on the 1-phosphoryl group of the substrate so that nucleophilic attack on the 1-phosphorus atom is facilitated. In addition, the monovalent cations may, along with Mg2+ ions and surrounding residues of the protein, help orient the 1-phosphoryl group so as to achieve the optimal position required for catalysis. Monovalent cations inhibit FBPase either by distorting the geometry of the active site or by retarding turnover or product release.

Metrifonate induces cholinesterase inhibition exclusively via slow release of dichlorvos

Abstract: Metrifonate, a long-acting cholinesterase (ChE) inhibitor with very low toxicity in warm-blooded animals, inhibits rat brain and serum cholinesterase (ChE) in vitro through its hydrolytic degradation product, dichlorvos. This conclusion is based on the finding that metrifonate-induced ChE inhibition showed the same pH dependence as its reported dehydrochlorination to dichlorvos. The ChE inhibition induced by dichlorvos was not pH dependent. It was mediated by a competitive drug interaction with the catalytic site of the enzyme, which led to irreversible inhibition within several minutes of incubation. After this time, addition of further substrate to the inhibited enzyme was not able to promote drug dissociation and hence enzyme reactivation. Similar characteristics of inhibition, i.e. interaction with the substrate binding site and time-dependent switch to non-competitive inhibition were observed with the reference compound, physostigmine. However, the physostigmine-induced inhibition of ChE could be readily reversed by further substrate addition. Another reference compound, tetrahydroaminoacridine (THA), also induced a reversible inhibition of rat brain and serum cholinesterase, but with a mechanism of action different from that of both dichlorvos and physostigmine in that enzyme inhibition occurred rapidly upon drug addition at an allosteric site on the enzyme surface. It is suggested that the unique slow release plus the slow inhibition of ChE by dichlorvos is responsible for the lower toxicity of metrifonate compared to that of directly acting ChE inhibitors.

Structure-function relationship of the inhibition of the 3,5,3'-triiodothyronine binding to the alpha1- and beta1-thyroid hormone receptor by amiodarone analogs.

Abstract: Desethylamiodarone (DEA), the major metabolite of the potent antiarrhythmic drug amiodarone (A), acts as a competitive inhibitor of T3, binding to the alpha1-thyroid hormone receptor (alpha1-T3R), but as a noncompetitive inhibitor with respect to the beta1-T3R. To gain insight into the structure- function relationship of the interaction between A metabolites and T3Rs, we investigated the effects of several A analogs on T3 binding to the alpha1-T3R and beta1-T3R in vitro. The analogs tested were: 1) compounds obtained by deethylation of A, DEA, and desdiethylamiodarone (DDEA); 2) compounds obtained by deiodination of A, monoiodoamiodarone and desdiiodoamiodarone (DDIA); and 3) benzofuran derivatives with various iodination grades, 2-butyl-3-(4-hydroxy-3,5-diiodo-benzoyl)benzofuran (L3373, two iodine atoms), L6424 (L3373 with one iodine atom), and L3372 (L3373, no iodine atoms). IC50, values of inhibition of T3 binding to alpha1-T3R and beta1-T3R, respectively, were as follows (mean +/- SD, expressed x 10(-5) M): DEA, 4.7 +/- 0.9 and 2.7 +/- 1.4 (P < 0.001); DDEA, 3.7 +/- 0.9 and 1.9 +/- 0.3 (P < 0.001); monoiodoamiodarone, more than 20 and more than 20; DDIA, 16.2 +/- 5.6 and 9.1 +/- 2.1 (P < 0.01); L3373, 3.8 +/- 1.0 and 3.6 +/- 0.5 (P = NS); L6424, 11.3 +/- 5.7 and 10 +/- 2.0 (P = NS); and L3372, no inhibition. Scatchard analyses in the presence of DDEA, DDIA, and L3373 demonstrated a dose-dependent decrease in Ka, but no change in the maximum binding capacity (MBC) of T3 binding to alpha1-T3R. Langmuir plots clearly indicated competitive inhibition of T3 binding to alpha1-T3R by DDEA, DDIA, and L3373. In contrast, these three analogs acted differently with respect to the beta1-T3R. DDEA and DDIA decreased both Ka and MBC in Scatchard plots using beta1-T3R, demonstrating noncompetitive inhibition. L3373 decreased dose-dependently Ka, but not MBC, values of T3 binding to the beta1-T3R and clearly acted as a competitive inhibitor. Ki plots indicated that DDEA, DDIA, and L3373 do not interfere significantly with occupied T3Rs. KI (inhibition constant for the unoccupied receptor) plots demonstrated increasing inhibition of the T3 binding to unoccupied receptors with increasing analog concentrations. In summary, 1) removal of one or two ethyl groups of A results in compounds with strong but almost equal potency of inhibiting T3R binding, whereas removal of one or two iodine atoms of A has a lower potency in this respect. The strong inhibitory potency of the benzofuran derivative L3373 (equalling that of the deethylated compounds) is lost upon deiodination. 2) All tested A analogs acted as competitive inhibitors to the alpha1-T3R. The behavior to the beta1-T3R was different; deethylation or deiodination of A resulted in noncompetitive inhibition, whereas L3373 was a competitive inhibitor. The potency of deethylated and deiodinated compounds (but not of the benzofuran derivatives) for inhibiting T3 binding was twice as high for the beta1-T3R as for the alpha1-T3R. 3) All tested A analogs preferentially interfere with T3 binding to unoccupied receptors. The implications of these findings for the structure-activity relationship are the following: 1) the size of the diethyl-substituted nitrogen group and of the two bulky iodine atoms in the A molecule hamper the binding of A at the T3 binding site of T3Rs; and 2) differences in the hormone-binding domain of alpha1- and beta1-T3Rs are likely to account for the competitive or noncompetitive nature of inhibition of T3 binding by A analogs.

Incorporation of an unnatural amino acid in the active site of porcine pancreatic phospholipase A2. Substitution of histidine by l,2,4-triazole-3-alanine yields an enzyme with high activity at acidic pH

Abstract: The effect of the substitution of the active site histidine 48 by the unnatural 1,2,4-triazole-3-alanine (TAA) amino acid analogue in porcine pancreas phospholipase A2 (PLA2) was studied. TAA was introduced biosynthetically using a his-auxotrophic Escherichia coli strain. To study solely the effect of the substitution of the active site histidine, two nonessential histidines (i.e. His17 and His115) were replaced by asparagines, resulting in a fully active mutant enzyme (His-PLA2). In this His-PLA2 the single histidine as position 48 was substituted by TAA with an incorporation efficiency of about 90%, giving a mixture of His-PLA2 and TAA-PLA2. Based on the charge difference at acidic pH, both forms could be separated by FPLC, allowing for the purification of TAA-PLA2 free from His-PLA2. At pH 6, TAA-PLA2 has a fivefold reduced activity compared with His-Pla2. This reduced activity paralells a reduced rate of covalent modification with p-nitrophenacyl bromide of TAA-PLA2 compared with His-PLA2. Competitive inhibition gave comparable IC50 values for WT-PLA2, His-PLA2 and TAA-PLA2. These results indicate that the reduction in activity is not caused by a different affinity for the substrate, but more likely results from a reduced kcat value in TAA-PLA2. The enzymatic activities for native and mutant PLA2s were measured at different pH values. For WT-PLA2 and His-PLA2 the activity is optimal at pH 6 and is strongly deminished at acidic pH, with no observable activity at pH 3. In contrast, TAA-PLA2 is as active at pH 3 as at pH 6. Most likely, the decrease in activity observed for WT-PLA2 and His-PLA2 is caused by the protonation of the active site His48, which is the general base involved in the activation of the nucleophilic water molecule. In TAA-PLA2, however, the active site residue TAA48 is unprotonated at both pH 3 and 6 as a result of the low pKa of TAA compared with histidine.

Expression, purification and characterization of recombinant phosphomannomutase and GDP-α-D-mannose pyrophosphorylase from Salmonella enterica, group B, for the synthesis of GDP-α-D-mannose from D-mannose

Abstract: The genes rfbK and rfbM from the rfb cluster (O-antigen biosynthesis) of Salmonella enterica, group B, encoding for the enzymes phosphomannomutase (EC 5.4.2.8) and GDP-alpha-D-mannose pyrophosphorylase (EC 2.7.7.13) were overexpressed in E.coli BL21 (DE3) with specific activities of 0.1 U/mg and 0.3-0.6 U/mg, respectively. Both enzymes were partially purified to give specific activities of 0.26 U/mg and 2.75 U/mg, respectively. Kinetic characterization of the homodimeric (108 kDa) GDP-alpha-D-mannose pyrophosphorylase revealed a K(m) for GTP and mannose-1-P of 0.2 mM and 0.01 mM with substrate surplus inhibition constants (Kis) of 10.9 mM and 0.7 mM, respectively. The product GDP-alpha-D-mannose gave a competitive inhibition with respect to GTP (Ki 14.7 microM) and an uncompetitive inhibition with respect to mannose-1-P (Ki 115 microM). Both recombinant enzymes were used for repetitive batch synthesis of GDP-alpha-D-mannose staring from D-mannose and GTP. In three subsequent batches 581 mg (960 mumol) GDP-alpha-D-mannose was synthesized with 80% average yield. The overall yield after product isolation was 22.9% (329 mumol, 199 mg).

Molecular modelling for the design of chimaeric biomimetic dye-ligands and their interaction with bovine heart mitochondrial malate dehydrogenase.

Abstract: Molecular modelling and kinetic inhibition studies, as well as KD determinations by both difference-spectra and enzyme-inactivation studies, were employed to assess the ability of purpose-designed chimaeric biomimetic dyes (BM dyes) to act as affinity ligands for bovine heart L-malate dehydrogenase (MDH). Each BM dye was composed of two enzyme-recognition moieties. The terminal biomimetic moiety bore a carboxyl or a keto acid structure linked to the triazine ring, thus mimicking the substrate of MDH. The chromophore anthraquinone moiety remained unchanged and the same as that of the parent dye Vilmafix Blue A-R (VBAR), recognizing the nucleotide-binding site of MDH. The monochlorotriazine BM dyes did not inactivate MDH but competitively inhibited inactivation by the parent dichlorotriazine dye VBAR. Dye binding to MDH was accompanied by a characteristic spectral change in the range 500-850 nm. This phenomenon was reversed after titration with increasing amounts of NADH. When compared with VBAR, Cibacron Blue 3GA and two control non-biomimetic anthraquinone dyes, all BM dyes exhibited lower KD values and therefore higher affinity for MDH. The enzyme bound preferably to BM ligands substituted with a biomimetic aromatic moiety bearing an alpha-keto acid group and an amide linkage, rather than a monocarboxyl group. Thus the biomimetic dye bearing p-aminobenzyloxanilic acid as its terminal biomimetic moiety (BM5) exhibited the highest affinity (KD 1.3 microM, which corresponded to a 219-fold decrease over the KD of a control dye). BM5 displayed competitive inhibition with respect to both NADH (Ki 2.7 microM) and oxaloacetate (Ki 9.6 microM). A combination of molecular modelling and experimental studies has led to certain conclusions. The positioning of the dye in the enzyme is primarily achieved by the recognition and positioning of the nucleotide-pseudomimetic anthraquinone moiety. The hydrophobic groups of the dye provide the driving force for positioning of the ketocarboxyl biomimetic moiety. A match between the alternating polar and hydrophobic regions of the enzyme binding site with those of the biomimetic moiety is desirable. The length of the biomimetic moiety should be conserved in order for the keto acid to approach the enzyme active site and form charge-charge interactions.

Enzyme inhibition by allicin, the molluscicidal agent of Allium sativum L. (garlic)

Abstract: Allicin, a molluscicidal component of garlic inhibited the activity of acetylcholinesterase, lactic dehydrogenase and alkaline phosphatase in in vivo and in vitro exposure against Lymnaea acuminata. It was observed that succinic dehydrogenase activity in the nervous tissue of Lymnaea acuminata was increased in in vivo treatment whereas with in vitro exposure, allicin caused no significant change in succinic dehydrogenase activity. The inhibition kinetics of these enzymes indicates that allicin caused an uncompetitive inhibition of AChE and a competitive inhibition of LDH and alkaline phosphatase.

Kinetics of inhibition of alkaline phosphatase from green crab (scylla serrata) by n-bromosuccinimide

Abstract: The inactivation of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide has been studied using the kinetic method of the substrate reaction during modification of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol.61, 381–436]. The results show that inactivation of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and is situated at the active site of the enzyme.