Differences in the interactions of liver alcohol dehydrogenases with probes binding into the substrate pocket

The interactions of three groups of probes (berberine alkaloids, tricyclic psychopharmaca and acridine derivatives) with isoenzymes of horse liver alcohol dehydrogenase and with rat liver alcohol dehydrogenase have been examined. These compounds inhibit the activity of the EE isoenzyme of horse liver alcohol dehydrogenase but differ in their behaviour towards the steroid-active enzymes (i.e. the ES isoenzyme of horse liver alcohol dehydrognase and alcohol dehydrogenase from rat liver): psychopharmaca inhibit, acridines activate and berberines do not bind. The ligands differ also in their influence on the modification of the EE isoenzyme by iodoacetate. Polarities (expressed as Kosower's Z values) of the respective binding sites on the EE isoenzyme were estimated from optical properties of bound probes. Berberines bind into a very hydrophobic area of the enzyme molecule, the binding site for psychopharmaca is moderately hydrophobic and that for acridines is rather polar. Steric arrangements of the binding sites are also discussed. The data presented confirm the existence of three distinct binding sites for these ligands in the substrate pocket of liver alcohol dehydrogenase.     

Studies of yeast alcohol dehydrogenase with 3-aminopyridine mononucleotide

Summary 3-Aminopyridine mononucleotide, a nicotinamide mononucleotide analog, was prepared by enzymatic cleavage of 3-aminopyridine adenine dinucleotide by a snake venom phosphodiesterase and isolated by means of ion exchange chromatography. The spectrophotometric and fluorometric properties of this analog were studied. Several anions were shown to quench the fluorescence intensity of this analog. pH was shown to have a pronounced effect on the fluorescence intensity. 3-Aminopyridine mononucleotide was shown to be a coenzyme-competitive inhibitor of yeast alcohol dehydrogenase. The 3-aminopyridine mononucleotide was diazotized with the use of nitrous acid. A time dependent irreversible inactivation of yeast alcohol dehydrogenase resulted from incubation with the diazotized 3-aminopyridine mononucleotide at pH 7.0. Incubation of the enzyme with NAD prior to the addition of the diazotized 3-aminopyridine mononucleotide protected the enzyme against inactivation.Recently, 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP), NAD and NADP analogs respectively, were synthesized by either chemical or enzymatic processes. The chemical, spectrophotometric properties of these dinucleotides have also been reported. It was demonstrated that these nucleotides serve as coenzyme-competitive inhibitors of dehydrogenases but did not function as coenzymes for oxidation-reduction reactions catalyzed by these enzymes. The pyridine amino group of AAD was diazotized and the diazotized derivative was shown to inactive yeast alcohol dehydrogenase irreversibly. Isolation of modified cysteine residue from the modified yeast alcohol dehydrogenase resulting from inactivation by diazotized AAD has been reported. Thus, diazotized AAD proved to be a site specific label for the coenzyme binding site of yeast alcohol dehydrogenase. It was of interest to prepared and determine the properties of a NMN analog, 3-aminopyridine mononucleotide (APMN). The preparation of APMN was accomplished by enzymatic cleavage of AAD with snake venom phosphodiesterase according to a method previously reported. This report deals with the preparation, properties and studies of APMN with yeast alcohol dehydrogenase.This work was supported in part by Research Grant GR-IX from Old Dominion University Research Foundation.     

Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide

Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett. 40, 29-32). Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label. The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site. The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants. In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified. Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.     

Kinetic characterization of yeast alcohol dehydrogenases. Amino acid residue 294 and substrate specificity

A three-dimensional model of yeast alcohol dehydrogenase, based on the homologous horse liver enzyme, was used to compare the substrate binding pockets of the three isozymes (I, II, and III) from Saccharomyces cerevisiae and the enzyme from Schizosaccharomyces pombe. Isozyme I and the S. pombe enzyme have methionine at position 294 (numbered as in the liver enzyme, corresponding to 270 in yeast), whereas isozymes II and III have leucine. Otherwise the active sites of the S. cerevisiae enzymes are the same. All four wild-type enzymes were produced from the cloned genes. In addition, oligonucleotide-directed mutagenesis was used to change Met-294 in alcohol dehydrogenase I to leucine. The mechanisms for all five enzymes were predominantly ordered with ethanol (but partially random with butanol) at pH 7.3 and 30 degrees C. The wild-type alcohol dehydrogenases and the leucine mutant had similar kinetic constants, except that isozyme II had 10-20-fold smaller Michaelis and inhibition constants for ethanol. Thus, residue 294 is not responsible for this difference. Apparently, substitutions outside of the substrate binding pocket indirectly affect the interactions of the alcohol dehydrogenases with ethanol. Nevertheless, the substitution of methionine with leucine in the substrate binding site of alcohol dehydrogenase I produced a 7-10-fold increase in reactivity (V/Km) with butanol, pentanol, and hexanol. The higher activity is due to tighter binding of the longer chain alcohols and to more rapid hydrogen transfer.     

The guanosine 3',5'-bis(diphosphate) (ppGpp) cycle. Comparison of synthesis and degradation of guanosine 3',5'-bis(diphosphate) in various bacterial systems.

4-(3-Bromoacetylpyridinio)butyldiphosphoadenosine was synthesized with a [carbonyl-14C]acetyl label. The reactive coenzyme analogue inactivates alcohol dehydrogenase from Bacillus stearothermophilus by forming a covalent enzyme-coenzyme compound. The inactivation kinetics as well as the spectral properties of the modified enzyme after treatment with sodium hyposulphite suggest that the analogue is bound at the coenzyme binding site. B. stearothermophilus alcohol dehydrogenase modified with 14C-labelled coenzyme analogue and subseqeuntly carboxymethylated with unlabelled iodoacetic acid was digested with trypsin. The radioactive peptide was isolated and sequenced in parallel with the corresponding peptide similarly isolated from unmodified enzyme that had instead been carboxymethylated with iodo[14C]acetic acid. Amino acid and sequence analysis show that Cys-38 of the B. stearothermophilus alcohol dehydrogenase was modified by the reactive coenzyme analogue. This residue is homologous to Cys-43 in yeast alcohol dehydrogenase and Cys-46 in the horse liver enzyme but, unlike the latter two, Cys-38 is not reactive towards iodoacetate in the native bacterial enzyme.     

Subunit interactions in liver alcohol dehydrogenase: A partial alkylation study.

The transient kinetics of aldehyde reduction by NADH catalyzed by liver alcohol dehydrogenase consist of two kinetic processes. This biphasic rate behavior is consistent with a model in which one of the two identical subunits in the enzyme is inactive during the reaction at the adjacent protomer. Alternatively, enzyme heterogeneity could result in such biphasic behavior. We have prepared liver alcohol dehydrogenase containing a single major isozyme; and the transient kinetics of this purified enzyme are biphasic.Addition of two [¹⁴C]carboxymethyl groups per dimer to the two “reactive” sulfhydryl groups (Cys46) yields enzyme which is catalytically inactive toward alcohol oxidation. Alkylated enzyme, as initially isolated by gel filtration chromatography at pH 7·5, forms an NAD⁺-pyrazole complex. However, the ability to bind NAD⁺-pyrazole is rapidly lost in pH 8·75 buffer; therefore, our alkylated preparations, as isolated by chromatography at pH 8·75, are inactive toward NAD⁺-pyrazole complex formation. We have prepared partially inactivated enzyme by allowing iodoacetic acid to react with liver alcohol dehydrogenase until 50% of the NAD⁺-pyrazole binding capacity remains; under these reaction conditions one [¹⁴C]carboxymethyl group is added per dimer. This partially alkylated enzyme preparation is isolated by gel filtration and has been aged sufficiently to lose NAD⁺-pyrazole binding ability at alkylated subunits. When solutions of native liver alcohol dehydrogenase and partially alkylated liver alcohol dehydrogenase containing the same number of unmodified active sites are allowed to react with substrate under single turnover conditions, partially alkylated enzyme is only half as reactive as native enzyme. This indicates that some molecular species in partially alkylated liver alcohol dehydrogenase that react with pyrazole and NAD⁺ during the active site titration do not react with substrate. These data are consistent with a model in which a subunit adjacent to an alkylated protomer in the dimeric enzyme is inactive toward substrate. In addition, NAD⁺-pyrazole binding at the protomers adjacent to alkylated subunits is slowly lost so that 75% of the enzyme-NAD⁺-pyrazole binding capacity is lost in 50% alkylated enzyme. These data supply strong evidence for subunit interactions in liver alcohol dehydrogenase.Binding experiments performed on partially alkylated liver alcohol dehydrogenase indicate that coenzyme binding is normal at a subunit adjacent to an alkylated protomer even though active ternary complexes cannot be formed. One hypothesis consistent with these results is the unavailability of zinc for substrate binding at the active site in subunits adjacent to alkylated protomers in monoalkylated dimer.     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

Spectroscopic investigation of binary and ternary coenzyme complexes of yeast alcohol dehydrogenase.

Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.     

Affinity chromatography of enzyme cofactors: the separation of NAD on immobilised dehydrogenase colums.

1. Alcohol dehydrogenase (EC 1.1.1.1.) has been immobilised to aminoethyl-cellulose by glutaraldehyde, to DEAE-cellulose by an s-triazine derivative and to agarose using CNBr. Lactate dehydrogenase has been immobilised to the latter two supports. 2. Their use for affinity chromatography of NAD was compared and alcohol dehydrogenase immobilised to CNBr-activated agarose chosen for detailed study due to the efficient coupling of applied enzyme and the specific nature of binding. 3. The efficiency of coupling of alcohol dehydrogenase dropped from 94.5 to 72.2% when the applied load was increased from 18 to 54 mg/g activated agarose. Activity relative to free enzyme fell from 21 to 11%. The binding of NAD was maximal between pH 5.5 and 6. With the lowest loading of enzyme, NAD binding fell from 450 to 320 mug/g support when the linear flow rate was increased from 0.84 to 3.95 cm/min. 4. NAD was completely separated from a mixture with ATP, ADP and AMP. Separation from NMN and hydrolysed RNA and DNA was evidently possible. Immobilised alcohol dehydrogenase used for 34 binding experiments over a period of weeks maintained 60% of its original enzyme activity. 5. The method was applied to yeast NAD following mechanical disruption of yeast, clarification and either ultrafiltration or hollow-fibre dialysis to permit separate purification of macromolecules and nucleotides.     

Potential cancer chemopreventive activity of simple isoquinolines, 1-benzylisoquinolines, and protoberberines

Seventeen simple isoquinolines, 15 1-benzylisoquinolines, and 19 protoberberines were tested for their inhibitory activities against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among the tested alkaloids, the inhibitory activity of all 1-benzylisoquinolines and 11 protoberberines was higher than that of beta-carotene. The 1-benzylisoquinolines 19, 21, 22, 29, and 34 and protoberberines 41, 47-49, 51, 52, and 55 showed potent inhibitory effects on EBV-EA induction (96-100% inhibition at 1 x 10(3) mol ratio/TPA). These alkaloids were more active than the naturally occurring alkaloids, 23, 25, 33, 53, and 54. In addition, fifteen simple isoquinolines, eighteen 1-benzylisoquinolines and eight protoberberines were evaluated with respect to their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. Nine simple isoquinolines, ten 1-benzylisoquinolines, and four protoberberines were more potent than alpha-tocopherol, and four 1-benzylisoquinolines, 20 and 28-30, exhibited potent activities (SC50 4.5-5.8 microM). Their activities were higher than the naturally occurring alkaloids, 23, 25, and 33. Therefore, some of the isoquinoline alkaloids indicating the high activity on both assays may be potentially valuable cancer chemopreventive agents. Structure-activity relationships are discussed for both tests.     

Glutathione-independent Formaldehyde Dehydrogenase from Pseudomons putida: Survey of Functional Groups with Special Regard for Cysteine Residues

The role of cysteine residues for structure and function of formaldehyde dehydrogenase from Pseudomonas putida was analysed by amino acid sequence comparison, homology-based structure modeling, site-directed mutagenesis, and chemical modification. Five out of seven cysteine residues found in the enzyme were concluded to coordinate with an active site zinc (Cys-46) and structural zinc atoms (Cys-97, -100, -103, and -111) from the sequence comparison with other Zn-containing medium-chain alcohol dehydrogenase homologues. The three-dimensional structure model based on the known structure of the horse liver E-type alcohol dehydrogenase (ADH) indicated that Cys-257 is located very far from the active site Zn and NAD⁺ binding region, suggesting that Cys-257 does not participate in the enzyme reaction. The structure also suggested that Cys-166 does not coordinate to active site Zn, but Asp-169 functions as a Zn-ligand, instead.     

Human glutathione-dependent formaldehyde dehydrogenase. Structures of apo,binary, and inhibitory ternary complexes

The human glutathione-dependent formaldehyde dehydrogenase is unique among the structurally studied members of the alcohol dehydrogenase family in that it follows a random bi bi kinetic mechanism. The structures of an apo form of the enzyme, a binary complex with substrate 12-hydroxydodecanoic acid, and a ternary complex with NAD+ and the inhibitor dodecanoic acid were determined at 2.0, 2.3, and 2.3 A resolution by X-ray crystallography using the anomalous diffraction signal of zinc. The structures of the enzyme and its binary complex with the primary alcohol substrate, 12-hydroxydodecanoic acid, and the previously reported binary complex with the coenzyme show that the binding of the first substrate (alcohol or coenzyme) causes only minor changes to the overall structure of the enzyme. This is consistent with the random mechanism of the enzyme where either of the substrates binds to the free enzyme. The catalytic-domain position in these structures is intermediate to the "closed" and "open" conformations observed in class I alcohol dehydrogenases. More importantly, two different tetrahedral coordination environments of the active site zinc are observed in these structures. In the apoenzyme, the active site zinc is coordinated to Cys44, His66 and Cys173, and a water molecule. In the inhibitor complex, the coordination environment involves Glu67 instead of the solvent water molecule. The coordination environment involving Glu67 as the fourth ligand likely represents an intermediate step during ligand exchange at the active site zinc. These observations provide new insight into metal-assisted catalysis and substrate binding in glutathione-dependent formaldehyde dehydrogenase.     

Dye affinity labelling of yeast alcohol dehydrogenase

The interaction of yeast alcohol dehydrogenase (ADH) with the reactive chlorotriazine dye Vilmafix Blue A-R (VBAR) was studied. VBAR was purified to homogeneity on lipophilic Sephadex LH-20 and characterised by reverse phase HPLC and analytical TLC. Incubation of ADH with purified VBAR at pH 8.0 and 37 degrees C resulted in a time-dependent inactivation of the enzyme. The observed rate of enzyme inactivation (kobs) exhibited a non-linear dependence on VBAR concentration from 22 to 106 nmol, with a maximum rate of inactivation (k3) of 0.134 min-1 and kD of 141.7 microM. The inhibition was irreversible and activity could not be recovered by gel-filtration chromatography. The inactivation of ADH by VBAR was competitively inhibited by the nucleotides NADH and NAD+. These results suggest that VBAR acts as an affinity label at the nucleotide binding site of yeast ADH.     

Inhibition of DNA topoisomerase I by natural and synthetic mono- and dimeric protoberberine alkaloids

A series of natural (i.e., 1-7) and synthetic (i.e., 8-23) protoberberine alkaloids were evaluated for their inhibitory activities towards DNA topoisomerase I. Both the natural, monomeric protoberberine alkaloids and their mono-modified congeners showed only minor activities. In contrast, most of the dimeric protoberberine alkaloids, especially compounds 12-22, were highly active, with a similar cleavage efficiency as camptothecin (CPT), a well-known, potent topoisomerase-I inhibitor. Thus, these dimeric compounds are promising candidates to be further elaborated as anticancer leads. The mechanism of topoisomerase-I inhibition seems to be dependent on drug concentration for the dimeric protoberberines. At low concentration, they exhibit similar characteristics as CPT. At high concentration, this ability is mostly lost, and the dimers inhibit the relaxation activity of topoisomerase I. Thus, we suppose that the active, dimeric protoberberines strongly bind to plasmid DNA at elevated drug concentration. This most likely results in blocking the enzyme's access to plasmid DNA, thus inhibiting its relaxation.     

Effect of alkaloids from celandine on calcium accumulation and oxidative phosphorylation in mitochondria depending on their DNA intercalating properties

The effect of principal alkaloids (sanguinarine, chelerythrine, coptisine, chelidonine) of greater celandine Chelidonium majus L., as well as the alkaloids from Colchicum autumnale L. (colchicine and colchamine) on calcium accumulation and oxidative phosphorylation in rat liver mitochondria has been studied. The obtained data were compared with DNA intercalating properties of alkaloids detected by the method of thermodenaturation (DNA melting curve plots). It was found that chelerythrine and sanguinarine blocked absorption and accumulation of calcium cations and inhibited oxidative phosphorylation, while the coptisine significantly diminished those indices. Chelidonine, colchicines and colchamine had no influence on the studied characteristics. The effect of alkaloids upon mitochondria functional state correlated tightly with their DNA intercalating properties: chelerythrine and sanguinarine were strong intercalators, while coptisine was a weak one, and chelidonine, colchicine and colchamine did not interact with DNA and caused no changes in its melting point. Correlation coefficient between the intercalating properties of alkaloids and their inhibition of calcium accumulation was 0.89, and with their oxidative phosphorylation inhibition - 0.93. It is suggested that the effect of studied alkaloids upon functional properties of mitochondria can be mediated by mtDNA.     

A two-step synthesis of new water-soluble polymers of NAD+ and ADP. The biological properties of these polymers

Alkylation at the N-1 position of the adenine moiety of NAD+, ADP or ATP with 2,3-epoxypropyl acrylate, followed by polymerization with or without acrylamide at pH 8, gave water-soluble polymers of NAD+ and ADP where the alkyl chain was located at the exocyclic adenine C-6 amino group. Cofactor incorporations were good to high: 145-447 mumol NAD+/g polymer and 667 mumol ADP/g polymer. About 30% of the bound NAD+ could be reduced with rabbit muscle lactae dehydrogenase, yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase; 84% of the bound ADP was phosphorylated with rabbit muscle creatine kinase. High cofactor activities were obtained with polymerized NAD+ with alcohol dehydrogenase as enzyme: the initial rate of NAD+ polymer reduction was 35-81% that of free NAD+. These values remained substantially high with agarose-immobilized alcohol dehydrogenase (15-36%) and should eventually allow their use in continuous enzymatic reactors. Enzymatic phosphorylation of ADP polymer by creatine kinase gave an ATP polymer with high biological activity: 480 mumol ATP/g polymer were transformed with yeast hexokinase.     

Identification of specific interactions that drive ligand-induced closure in five enzymes with classic domain movements

In order to better understand ligand-induced closure in domain enzymes, open unliganded X-ray structures and closed liganded X-ray structures have been studied in five enzymes: adenylate kinase, aspartate aminotransferase, citrate synthase, liver alcohol dehydrogenase, and the catalytic subunit of cAMP-dependent protein kinase. A sequential model of ligand binding and domain closure was used to test the hypothesis that the ligand actively drives closure from an open conformation. The analysis supports the assumption that each enzyme has a dedicated binding domain to which the ligand binds first and a closing domain. In every case, a small number of residues are identified to interact with the ligand to initiate and drive domain closure. In all cases except adenylate kinase, the backbone of residues located in an interdomain-bending region (hinge site) is identified to interact with the ligand to aid in driving closure. In adenylate kinase, the side-chain of a residue located directly adjacent to a bending region drives closure. It is thought that by binding near a hinge site the ligand is able to get within interaction range of residues when the enzyme is in the open conformation. Interdomain bending regions not involved in inducing closure are involved in control, helping to determine the location of the hinge axis. Similarities have been discovered between aspartate aminotransferase and citrate synthase that only come to light in the context of their dynamical behaviour in response to binding their substrate. Similarity also exists between liver alcohol dehydrogenase and cAMP-dependent protein kinase whereby groups on NAD and ATP, respectively, mimic the backbone of a single amino acid residue in a process where a three residue segment located at the terminus of a beta-sheet, moves to form hydrogen bonds with the mimic that resemble those found in a parallel beta-sheet. This interaction helps to drive domain closure in a process that has analogy to protein folding.     

RNA targeting by small molecules: Binding of protoberberine, benzophenanthridine and aristolochia alkaloids to various RNA structures

Studies on RNA targeting by small molecules to specifically control certain cellular functions is an area of remarkable current interest. For this purpose, a basic understanding of the molecular aspects of the interaction of small molecules with various RNA structures is essential. Alkaloids are a group of natural products with potential therapeutic utility, and very recently, their interaction with many RNA structures have been reported. Especially noteworthy are the protoberberines and aristolochia alkaloids distributed widely in many botanical families. Many of the alkaloids of these group exhibit excellent binding affinity to many RNA structures that may be exploited to develop RNA targeted therapeutics. This review attempts to present the current status on the understanding of the interaction of these alkaloids with various RNA structures, mainly highlighting the biophysical aspects.     

Alcohol dehydrogenase from the fruitfly Drosophila melanogaster. Inhibition studies of the alleloenzymes AdhS and AdhUF

Different metal binding inhibitors of horse liver alcohol dehydrogenase, similarly affect the Drosophila melanogaster AdhS and AdhUF alleloenzymes. However, binding is generally weaker and the experiments show that the alleloenzymes although not zinc metalloenzymes, behave to the metal binding reagents very much as if they were. The metal-directed, affinity-labelling, imidazole derivative BrImPpOH reversibly inhibits, but does not inactivate the alleolenzymes. This confirms there is no active site metal atom with cysteine as a metal ligand, as found in zinc alcohol dehydrogenases. Pyrazole is a strong ethanol-competitive inhibitor of AdhS and AdhUF alleloenzymes. Formation of the ternary enzyme-NAD-pyrazole complex gives an absorption increase between 295-330 nm. This enables an active site titration to be performed and the determination of epsilon (305 nm) of 15.8 . 10(3) M-1 . cm-1. Inhibition experiments with imidazole confirm that with secondary alcohols such as propan-2-ol, a Theorell-Chance mechanism predominates, but with ethanol and primary alcohols, interconversion of the ternary complexes is rate limiting. Salicylate is a coenzyme competitive inhibitor and KEI suggests that the coenzyme adenosine binding region is similar is Drosophila and horse liver alcohol dehydrogenase. Drosophila alcohol dehydrogenase is found not to form a ternary complex with NADH and isobutyramide. In this and other properties it is like carboxymethyl liver alcohol dehydrogenase. Both Drosophila and carboxymethyl alcohol dehydrogenase bind coenzyme in a similar manner to native horse liver alcohol dehydrogenase, but substrate binding differs between each. Inhibition by Cibacrone blue, indicates that amino acid 192 which is lysine in AdhS and threonine in AdhUF, is located in the coenzyme-binding region. Proteolytic activity present in preparations of alcohol dehydrogenase from D. melanogaster, is considered due to a metalloprotease, for which BrImPpOH is a potent inactivator.