Autoradiographical distribution of imidazoline binding sites in monoamine oxidase A deficient mice

This study has used receptor autoradiography to characterize imidazoline binding sites (I-BS) in monoamine oxidase (MAO) A knockout and wild-type mice. A comparison between MAO-A and MAO-B, binding of the endogenous beta-carboline [(3)H]harmane, and I-BS, has been made using sections from brain and kidney. The loss of binding to MAO-A in the knockout animals was confirmed using the selective radioligand [(3)H]Ro41-1049, with labelling reduced to background levels. The binding of [(3)H]Ro19-6327 to MAO-B was unaffected, indicating no change in this isoform in response to the loss of MAO-A. A reduction in binding to the I(2)-BS, as labelled by both [(3)H]idazoxan and [(3)H]2-BFI (2-(2-benzofuranyl)-2-imidazoline), was seen in the MAO-A knockout animals in both brain and kidney sections, whereas binding to the I(1)-BS in kidney sections remained unchanged. The loss of I(2) binding was found to be regionally dependent and was positively correlated with the relative expression of MAO-A in specific regions in the wild-type animals. Using the MAO-A knockout mice it was also possible to demonstrate a non-MAO-A population of binding sites labelled by the putative I-BS endogenous ligand, harmane.     

Novel monoamine oxidase inhibitors based on the privileged 2-imidazoline molecular framework

Series of structurally diverse 2-imidazoline derivatives have been synthesized by condensation of substituted aldehydes with ethylenediamine, Pd-catalyzed N-arylation of 2-imidazolines and by the formation of 1,2,4-oxadiazoles and benzoxazepines from 2-imidazoline-containing precursors. The 2-imidazoline derivatives were evaluated as potential inhibitors of human monoamine oxidase (MAO) A and B. Among the 2-imidazolines, good potency inhibitors were discovered with compound 9p (IC₅₀?=?0.012?µM) being the most potent MAO-B inhibitor, while compound 9d (IC₅₀?=?0.751?µM) was the most potent MAO-A inhibitor of the series. These potencies are in the same range as those of reference MAO inhibitors used in the clinic. Among 33 compounds evaluated, 13 exhibited IC₅₀ values in the submicromolar range for the inhibition of an MAO isoform. It is postulated that the imidazoline moieties of some of these inhibitors may be recognized by the imidazoline I₂-binding site of MAO. Good potency MAO inhibitors may be useful for the treatment of neuropsychiatric and neurodegenerative disorders such as depression and Parkinson’s disease, and future application for the treatment of prostate cancer, congestive heart failure and Alzheimer’s disease. In addition, high potency 2-imidazoline-derived MAO inhibitors may be used as potential probes for the imidazoline binding sites of the MAOs, as well as to determine alternative binding regions of imidazoline within the MAO active site.     

Targeting imidazoline site on monoamine oxidase B through molecular docking simulations

Monoamine oxidase (MAO) is an enzyme of major importance in neurochemistry, because it catalyzes the inactivation pathway for the catecholamine neurotransmitters, noradrenaline, adrenaline and dopamine. In the last decade it was demonstrated that imidazoline derivatives were able to inhibit MAO activity. Furthermore, crystallographic studies identified the imidazoline-binding domain on monoamine oxidase B (MAO-B), which opens the possibility of molecular docking studies focused on this binding site. The goal of the present study is to identify new potential inhibitors for MAO-B. In addition, we are also interested in establishing a fast and reliable computation methodology to pave the way for future molecular docking simulations focused on the imidazoline-binding site of this enzyme. We used the program 'molegro virtual docker' (MVD) in all simulations described here. All results indicate that simplex evolution algorithm is able to succesfully simulate the protein-ligand interactions for MAO-B. In addition, a scoring function implemented in the program MVD presents high correlation coefficient with experimental activity of MAO-B inhibitors. Taken together, our results identified a new family of potential MAO-B inhibitors and mapped important residues for intermolecular interactions between this enzyme and ligands.     

Imidazoline Receptor Contributes to Ion and Water Transport across the Intestine of the Eel Acclimated to Sea Water

Guanabenz, an I2-imidazoline-related compound with high affinity for intestinal membrane of the eel (), enhanced the transepithelial potential difference (PD) and short-circuit current (Isc) from serosa to mucosa after pretreatment with isobutylmethylxanthine (IBMX), serotonin (5-HT) and methacholine (MCh). The mucosal effect of guanabenz was not mimicked by adrenaline, indicating that the mucosal guanabenz binding site is not adrenoceptors. The mucosal guanabenz enhanced the Isc in a concentration-dependent manner. Similar enhancement in the Isc was also obtained after addition of other imidazoline derivatives such as ST93, clonidine, ST91, naphazoline and UK14,304 into the mucosal fluid. On the other hand, the effect of guanabenz was completely blocked by mucosal RX821002 or efaroxan, another imidazoline derivatives. Since some imidazoline derivatives act as agonists and others as antagonist, there must exist imidazoline receptor on the mucosal side of the eel intestine. Accompanied by an increase in the PD, NaCl and water absorption across the intestine was also enhanced by mucosal guanabenz. To search for endogenous ligands for the imidazoline receptor, luminal fluid in the intestine of the seawater eels was collected. However, most luminal fluid was ineffective. Only one among 10 samples showed guanabenz-like activity, suggesting that the endogenous ligands is secreted into the lumen under restricted condition alone.     

Identification and characterization of the imidazoline I2b-binding sites in the hamster brown adipose tissue as a study model for imidazoline receptors

The imidazoline-type compound, MPV-1743, has been found to activate nonshivering thermogenesis (NST) in brown adipose tissue (BAT) of the genetically obese Zucker rats. The regulation of NST in BAT is linked to the catecholamine metabolism, and the imidazoline I2-binding sites have been found on the monoamine oxidase, a catecholamine metabolising enzyme. In this study, the I2-binding sites of hamster BAT have been characterised using a receptor binding assay with 3H-idazoxan as a radioligand, and the interaction of MPV-1743 with these I2-binding sites has been studied using the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089. Cirazoline was used to determine the specific binding of 3H-idazoxan to the imidazoline I2-binding sites. Rauwolscine was added in the 3H-idazoxan binding assay in order to inhibit any binding to potential alpha2-adrenergic sites. In the presence of rauwolscine mask 3H-Idazoxan labelled a population of non-adrenergic binding sites expressing the properties of the imidazoline I2b-receptor subtype similar to that found in the rat liver (cirazoline > guanabenz = amiloride > clonidine). The binding of 3H-idazoxan to the I2b-binding sites could be displaced by the imidazole compounds with the following affinities: detomidine (KiHigh 9.2 nM; KiLow 3200 nM), MPV-2088 (KiHigh 19 nM; IKiLow 760 nM) and MPV-2089 (KiHigh 190 nM; KiLow 1300 nM), atipamezole (3500 nM) and dexmedetomidine (Ki 8400 nM). These results have shown that the hamster BAT contains the imidazoline I2b-binding sites with heterogeneous binding properties for some test compounds. In addition, the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089, had high affinity to these BAT imidazoline I2b-binding sites. Therefore, it is suggested that the regulation of NST in the hamster BAT may be an attractive model to study the role of imidazoline I2b-binding sites.     

Synthesis and pharmacological evaluation of imidazoline sites I1 and I2 selective ligands

Several series of 2-aryl or heterocyclic-imidazoline compounds have been prepared and evaluated in vitro as imidazoline sites (I1 and I2) and alpha-adrenergic (alpha1 and alpha2) receptor ligands. Their pKi values indicate that linkage of the imidazoline moiety at the 2-position with an aromatic substituent dramatically decreases alpha-adrenergic affinity. I1 sites are more accessible by phenyl imidazolines substituted by a methyl or a methoxy group at the ortho or meta position. Indeed, 2-(2'-methoxyphenyl)-imidazoline (17) is one of the best I1 ligands ever reported (pKi = 8.53 and I1/I2 > 3388). On the other hand, I2 selectivity increases in the presence of a methyl group in the para position. The original compound, 2-(3'-fluoro-4'-tolyl)-imidazoline (31) is a new potent ligand for the I2 sites with high selectivity (pKi = 8.53 and I2/I1 > 3388).     

Significance of multiple forms of brain monoamine oxidase in situ as probed by electron spin resonance.

Spin-labeled hydroxyamphetamine, a competitive reversible inhibitor of brain monoamine oxidase, has been shown to be useful as an electron spin resonance (ESR) probe of the microenvironment of the active sites of the possible monoamine oxidase multiple forms. The ESR spectrum of spin-labeled hydroxyamphetamine was strongly quenched upon binding to the enzyme. The conformation of the active site of rat brain monoamine oxidase existing in various physical states, i.e. monoamine oxidase in situ (intact brain mitochondria), crude solubilized monoamine oxidase (MAOS) and isolated monoamine oxidase fractions (MAOa and MAOb) were critically and systematically examined. Nonlinear least squares regression analyses have been used to fit the binding data (obtained at room temperature with varying spin-labeled hydroxyamphetamine concentrations) to three groups of independent noninteracting ligand-binding models. A Gibbs-Helmholtz relationship was applied to the interpretation of the measured apparent association constant K as a function of temperature ranging from 4-50 degrees with increments of 2 degreesmfrom the extracted intensive parameters, k (intrinsic association constant) and deltaF (intrinsic free energy), as well as the apparent heat, deltaH, it was clear that the microenvironment of the binding sites existing in the more purified enzyme fractions MAOa and MAOb were similar to those found in the crude solubilized enzyme. More importantly, they correlated well with the conformation of the sites characterized in situ. The data suggested that the microenvironment of this multienzyme system was unperturbed in spite of the treatment due to the isolation process. In terms of the composition of binding sites, MAOa appeared to be heterogeneous while MAOb appeared to be more homogeneous. Since the isolated fractions MAOa and MAOb possessed marked different substrate specificities, these observations directly implied that monoamine oxidase multiple forms do exist in situ. The extracted extensive parameters, n (specific binding activity, nanomoles/mg of protein), as well as the measured characteristic transition temperatures, indicated that the relative abundance of the sites which directly affected substrate specificities was indeed altered. The consistency of the characteristic transition temperatures of 21 degrees and 38 degrees for the case of intact membrane preparations was particularly significant. A tenable hypothesis is that the manipulation in the composition of the monoamine oxidase binding forms through intimate lipid-protein interactions, which has been amply demonstrated in many biomembrane systems to be functionally important might be the underlying regulatory mechanism in vivo.     

Oxygen activation in flavoprotein oxidases: the importance of being positive

The oxidation of flavin hydroquinones by O(2) in solution is slow, with second-order rate constants of ~250 M(-1) s(-1). This is due to the obligatory, single-electron transfer that initiates the reaction being thermodynamically unfavored and poorly catalyzed. Notwithstanding considerations of O(2) accessibility to the reaction site, its desolvation and geometry and other factors that can also contribute to further rate acceleration, flavoprotein oxidases must activate O(2) for reaction with flavin hydroquinones to be able to achieve the 100-1000-fold rate enhancements typically observed. Protein positive charges have been identified in glucose oxidase, monomeric sarcosine oxidase, N-methyltryptophan oxidase and fructosamine oxidase that electrostatically stabilize the transition state for the initial single electron transfer that generates the O(2)(-?)/flavin semiquinone radical pair. In choline oxidase despite the presence of three histidines in the active site, the trimethylammonium group of the reaction product provides such an electrostatic stabilization. A nonpolar site proximal to the flavin C(4a) atom in choline oxidase has also been identified, which contributes to the geometry and desolvation of the O(2) reaction site. The relevance of O(2) activation by product charges to other flavoprotein oxidases, such as for example those catalyzing amine oxidations, is discussed in this review. A nonpolar site close to the flavin C(4a) atom and a positive charge is identified through structural analysis in several flavoprotein oxidases. Mutagenesis has disclosed nonpolar sites in O(2)-reducing enzymes that utilize copper/TPQ or iron. It is predicted that classes of O(2)-reducing enzymes utilizing other cofactors also contain a similar catalytic motif.     

Evidence for different pharmacological targets for imidazoline compounds inhibiting settlement of the barnacle Balanus improvisus

We describe the effect of eight different imidazoline/guanidinium compounds on the settlement and metamorphosis of larvae of the barnacle Balanus improvisus. These agents were chosen on the basis of their similar pharmacological classification in vertebrates and their chemical similarity to medetomidine and clonidine, previously described as highly potent settlement inhibitors (nanomolar range). Seven of the tested compounds were found to inhibit settlement in a dose-dependent manner in concentrations ranging from 100 nM to 10 microM without any significant lethal effects. In vertebrate systems these substances have overlapping functions and interact with both alpha-adrenoceptors as well as imidazoline binding sites. Antagonizing experiments using the highly specific alpha(2)-antagonist methoxy-idazoxan or agmatine (the putative endogenous ligand at imidazoline receptors) were performed to discriminate between putative pharmacological mechanisms involved in the inhibition of cyprid settlement. Agmatine was not able to reverse the effect of any of the tested compounds. However, methoxy-idazoxan almost completely abolished the settlement inhibition mediated by guanabenz (alpha(2)-agonist, I(2) ligand), moxonidine (alpha(2)-agonist, I(1) ligand) and tetrahydrozoline (alpha-agonist, I(2) ligand). The actions of cirazoline (alpha(1)-agonist, I(2) ligand) BU 224 (I(2) ligand) and metrazoline (I(2) ligand) were not reversed by treatment with methoxy-idazoxan. These results suggest that the settlement inhibition evoked by the I(2) ligands and alpha(2)-agonists used in this study of the neurologically simple but well-organized barnacle larva is mediated through different physiological targets important in the overall settlement process.     

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study.

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.     

Inhibition of rat brainstem monoamine oxidase activity by CGP 6085 A

CGP 6085 A [4-(5,6-dimethyl-2-benzofuranyl)piperidine] HCl, a known serotonin inhibitor, also inhibits rat brainstem monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in both in vivo and in vitro experiments. Serotonin (5-HT) deamination by MAO-A is inhibited 35% at a dose of 100 mg/kg i.p. in vivo. Similar experiments show a maximal 20% decrease in phenylethylamine (PEA) deamination by MAO-B at a dosage of 30 mg/kg i.p. Over the range of 0.1 to 10 mg/kg i.p., CGP 6085 A decreases 5-HIAA levels in the brainstem. This in vivo inhibition of MAO activity is confirmed by in vitro experiments. In vitro studies in rat brainstem mitochondrial preparations show a dose-dependent, reversible, inhibition of MAO using tyramine as the substrate for the enzyme reaction. With an in vitro IC50 of 2-3 microM, the potency of CGP 6085 A is comparable to pargyline.     

[3H]Harman Binding Experiments. I: A Reversible and Selective Radioligand for Monoamine Oxidase Subtype A in the CNS of the Rat

Harman (1-methyl-beta-carboline) is an endogenous compound with neurotropic properties in rats and humans. In a novel in vitro binding assay, the binding site of [3H]harman has been characterized in the rat crude mitochondrial (P2) fraction. The binding was saturable and reversible. Only a single high-affinity binding site was detected by kinetic, saturation, and displacement analyses in the cerebral cortex of the rat. The linear Scatchard plots revealed equilibrium dissociation constant (KD) values of approximately 2.5 nM at 0 degrees C, approximately 9 nM at 23 degrees C, and approximately 30 nM at 37 degrees C. Among six CNS regions (hypothalamus, hippocampus, cerebral cortex, striatum, cerebellum, and spinal cord), the highest density of binding sites (Bmax) was determined in the hypothalamus (approximately 5.5 pmol/mg of protein) and the lowest in the spinal cord (approximately 2.0 pmol/mg of protein). Several drugs known to affect serotonergic, adrenergic, dopaminergic, cholinergic, or GABAergic neurotransmission inhibited specific binding at best in the micromolar range. In contrast, potent and selective inhibitors of monoamine oxidase subtype A were active in the lower and middle nanomolar range. The displacing potency (apparent Ki) of substrates and inhibitors of monoamine oxidase correlated positively and highly significantly with the corresponding values of the inhibition of monoamine oxidase activity of subtype A (r = 0.92, p less than 0.001, n = 17) but not of subtype B (r = -0.47, p greater than 0.05, n = 15). In conclusion, [3H]harman was identified as a specific ligand of the active site of the A subtype of monoamine oxidase in rat brain.     

Purification and properties of mitochondrial monoamine oxidase type A from human placenta

A high yield purification scheme for monoamine oxidase A from human placental mitochondria is described. The enzyme is solubilized by a combination of treatment with phospholipase A and C and extraction with Triton X-100 and further purified by partitioning between dextran and polyethylene glycol polymers. The enzyme was obtained in 35% yield and high purity on DEAE-Sepharose CL-6B chromatography. This product, 90% catalytically active, showed a single major and several minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further purification could be achieved by additional chromatography using Bio-Gel HTP, but concomitant loss of catalytic activity occurred (enzyme remained about 60% active). The difference extinction coefficient for flavinox--flavinred at 456 nm was 10,800 +/- 350 m-1 cm-1. A sulfhydryl to flavin ratio of 7.5 was obtained when enzyme was denatured with sodium dodecyl sulfate, reduced with 2-mercaptoethanol, and titrated with 2,2'-dipyridyl disulfide. Anaerobic titration with 0.5 eq of sodium dithionite gave rise to the red anionic flavin radical, and full reduction was observed on further addition of reagent. The Km value for kynuramine was essentially the same for mitochondria (0.12 mM) and enzyme after DEAE-Sepharose CL-6B chromatography (0.17 mM). The concentration of clorgyline and deprenyl required for 50% inactivation also remained essentially unchanged. Incubation of the enzyme with 2,2'-dipyridyl disulfide caused inactivation in a biphasic manner with apparent second-order rate constants of 1230 M-1 min-1 and 235 M-1 min-1 for the rapid and slow phase, respectively. This inactivation was largely abolished by the inclusion of the competitive inhibitor amphetamine (Ki = 20 microM) in the incubation mixture. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a subunit molecular mass of 60-64 kDa, about 1.5-2.5 kDa higher than human liver monoamine oxidase B.     

Molecular pharmacology of the catecholamine transporter of chromaffin granules from the bovine adrenal medulla

Tetrabenazine (TBZ) and reserpine are two inhibitors of the catecholamine uptake system of the chromaffin granule membrane. They are structural analogs of the substrates dopamine and serotonin and they inhibit the monoamine transporter, which catalyzes a H+/neutral amine antiport. [3H]Dihydrotetrabenazine ([3H]TBZOH) is bound by chromaffin granule membranes on one class of site (T sites, KD = 3 nM); [3H]reserpine is bound on T sites and a second class of site (R1 sites, KD = 0.7 nM). The two sites are involved in monoamine translocation. The substrates displace the ligands with different efficiency: noradrenaline (Km = 10 microM) displaces reserpine efficiently (EC50 = 30 microM), but TBZOH poorly (EC50 = 2000 microM); m-iodobenzylguanidine, which has recently been shown to be a substrate of the monoamine uptake system (Km = 5 microM), displaces TBZOH efficiently (EC50 = 25 microM), but reserpine inefficiently (EC50 = 300 microM). Since both substrates are translocated by the same transporter, this result confirms the existence of two sites with different properties. T sites are characterized by a linear relationship between the reciprocal of the dissociation constants of various drugs displacing [3H]TBZOH and their partition coefficient in octanol/H2O mixtures. This relationship, which indicates a hydrophobic environment of T sites, does not exist for R1 sites. T sites have been identified by covalent labeling with a derivative of TBZ coupled to an arylazido group. The labeled sites are borne by a 65,000 dalton protein. The kinetics of reserpine binding are accelerated in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of Ligand Binding through Cooperative Effects in Monoamine Oxidase B

Crystallographic and biochemical studies have been employed to identify the binding site and mechanism for potentiation of imidazoline binding in human monoamine oxidase B (MAO B). 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) inhibits recombinant human MAO B with a K_i of 8.3 ± 0.6 μm, whereas tranylcypromine-inhibited MAO B binds 2-BFI with a K_d of 9 ± 2 nm, representing an increase in binding energy Δ(ΔG) of −3.9 kcal/mol. Crystal structures show the imidazoline ligand bound in a site that is distinct from the substrate-binding cavity. Contributions to account for the increase in binding affinity upon tranylcypromine inhibition include a conformational change in the side chain of Gln²⁰⁶ and a “closed conformation” of the side chain of Ile¹⁹⁹, forming a hydrophobic “sandwich” with the side chain of Ile³¹⁶ on each face of the benzofuran ring of 2-BFI. Data with the I199A mutant of human MAO B and failure to observe a similar binding potentiation with rat MAO B, where Ile³¹⁶ is replaced with a Val residue, support an allosteric mechanism where the increased binding affinity of 2-BFI results from a cooperative increase in H-bond strength through formation of a more hydrophobic milieu. These insights should prove valuable in the design of high affinity and specific reversible MAO B inhibitors.     

The structure of the zinc sites of Escherichia coli DNA-dependent RNA polymerase.

X-ray absorption spectroscopy is ideally suited for the investigation of the electronic structure and the local environment (approximately 5 A) of specific atoms in biomolecules. While the edge region provides information about the valence state of the absorbing atom, the chemical identity of neighboring atoms, and the coordination geometry, the extended x-ray absorption fine structure region contains information about the number and average distance of neighboring atoms and their relative disorder. The development of sensitive detection methods has allowed studies using near physiological concentrations (as low as approximately 100 microM). RNA polymerase from Escherichia coli contains two zinc atoms: one tightly bound in the beta' subunit, the subunit that participates in template binding, and the other loosely bound in the beta subunit, the subunit that participates in substrate binding. X-ray absorption studies of these zinc sites in the native protein and of the zinc site in the beta' subunit after removal of the zinc in the beta subunit site by p-(hydroxymercuri)benzenesulfonate (Giedroc, D. P., and Coleman, J. E. (1986) Biochemistry 25, 4969-4978) indicate that both zinc sites have octahedral coordination. The zinc in the beta' subunit site has four sulfur ligands at an average distance of 2.36 +/- 0.02 A and two oxygen (or nitrogen) ligands at an average distance of 2.23 +/- 0.02 A. The beta subunit zinc site has five sulfur ligands at an average distance of 2.38 +/- 0.01 A and one histidine nitrogen ligand at 2.14 +/- 0.02 A. These results are in general agreement with earlier biochemical and spectroscopic studies.     

Time-resolved fluorescence studies of heterotropic ligand binding to cytochrome P450 3A4

Cytochrome P450 3A4 (CYP3A4) is a major enzymatic determinant of drug and xenobiotic metabolism that demonstrates remarkable substrate diversity and complex kinetic properties. The complex kinetics may result, in some cases, from multiple binding of ligands within the large active site or from an effector molecule acting at a distal allosteric site. Here, the fluorescent probe TNS (2-p-toluidinylnaphthalene-6-sulfonic acid) was characterized as an active site fluorescent ligand. UV-vis difference spectroscopy revealed a TNS-induced low-spin heme absorbance spectrum with an apparent K(d) of 25.4 +/- 2 microM. Catalytic turnover using 7-benzyloxyquinoline (7-BQ) as a substrate demonstrated TNS-dependent inhibition with an IC(50) of 9.9 +/- 0.1 microM. These results suggest that TNS binds in the CYP3A4 active site. The steady-state fluorescence of TNS increased upon binding to CYP3A4, and fluorescence titrations yielded a K(d) of 22.8 +/- 1 microM. Time-resolved frequency-domain measurement of TNS fluorescence lifetimes indicates a testosterone (TST)-dependent decrease in the excited-state lifetime of TNS, concomitant with a decrease in the steady-state fluorescence intensity. In contrast, the substrate erythromycin (ERY) had no effect on TNS lifetime, while it decreased the steady-state fluorescence intensity. Together, the results suggest that TNS binds in the active site of CYP3A4, while the first equivalent of TST binds at a distant allosteric effector site. Furthermore, the results are the first to indicate that TST bound to the effector site can modulate the environment of the heterotropic ligand.     

Inhibitors alter the spectrum and redox properties of monoamine oxidase A

Monoamine oxidase A (MAO A) catalyses the oxidation of both neurotransmitter and ingested amines. The mechanism of catalysis involves the covalently bound FAD cofactor. Although substrates and inhibitors alter the thermodynamic and kinetic properties of the flavin, how the ligands interact with the flavin is unknown. This work characterises the spectral changes that occur on inhibitor binding to MAO A and examines how the binding influences the flavin. The inhibitors, D-amphetamine, harmine, tetrindole, and befloxatone all induce similar (but not identical) changes in the spectrum of MAO A, consistent with stacking of inhibitor with the flavin in the active site. D-Amphetamine, harmine, and tetrindole stabilise the semiquinone form of FAD during reduction of MAO A by dithionite and no further reduction of these inhibitor-MAO A complexes has been observed. In contrast, semiquinone is never observed during reduction of the befloxatone-MAO A complex. Instead, partial reduction directly to the FADH(2) form occurs extremely slowly. Thus, inhibitor binding has a strong, structure-dependent influence on the environment of the flavin that alters its electronic properties.     

L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma. Comparative sequence analysis and characterization of active and inactive forms of the enzyme.

Here we report the cDNA-deduced amino-acid sequence of L-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FAD-dependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, D-amino-acid oxidases and tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl(-), prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.