Protein Ligand Interactions. Part 5: Isoquinoline Alkaloids as Inhibitors of Acetylcholinesterase from Electrophorus Electricus

Abstract

Kinetic analysis has shown that papaverine, berberine and isoquinoline alkaloids act as reversible competitive inhibitors of acetylcholinesterase with respect to the substrate, acetylthiocholine chloride. The inhibitor constants (Ki) vary from 3.5 μM to 88 μM. With time they act as irreversible covalent inhibitors with papaverine producing 85% inactivation after 40 min. Pseudo first-order kinetics are observed with the rate constant being proportional to the concentration of the ligand and the order of reaction being equal to one. Spectrophotometry was used to study the binding of the ligands with acetylcholinesterase and Scatchard analysis used to calculate the respective dissociation constants and the number of binding sites.     

Malate dehydrogenase, circular dichroism difference spectra of porcine heart mitochondrial and supernatant enzymes, binary enzyme-coenzyme, and ternary enzyme-coenzyme-substrate analog complexes.

Circular dichroism spectra and circular dichroism difference spectra, generated when porcine heart mitochondrial and supernatant malate dehydrogenase bind coenzymes or when enzyme dihydroincotinamide nucleotide binary complexes bind substrate analogs, are presented. No significant changes are observed in protein chromophores in the 200- to 240-nm spectral range indicating that there is apparently little or no perturbation of the alpha helix or peptide backbone when binary or ternary complexes are formed. Quite different spectral perturbances occur in the two enzymes with reduced coenzyme binding as well as with substrate-analog binding by enzyme-reduced coenzyme binding. Comparison of spectral perturbations in both enzymes with oxidized or reduced coenzyme binding suggests that the dihydronicotinamide moiety of the coenzyme interacts with or perturbs indirectly the environment of aromatic amino acid residues. Reduced coenzyme binding apparently perturbs tyrosine residues in both mitochondrial malate dehydrogenase and lactic dehydrogenase. Reduced coenzyme binding perturbs tyrosine and tryptophan residues in supernatant malate dehydrogenase. The number of reduced coenzyme binding sites was determined to be two per 70,000 daltons in the mitochondrial enzyme, and the reduced coenzyme dissociation constants, determined through the change in ellipticity at 260 nm, with dihydronicotinamide adenine dinucleotide binding, were found to be good agreement with published values (Holbrook, J. J., and Wolfe, R. G. (1972) Biochemistry 11, 2499-2502) obtained through fluorescence-binding studies and indicate no apparent extra coenzyme binding sites. When D-malate forms a ternary complex with malate dehydrogenase-reduced coenzyme complexes, perturbation of both adenine and dihydronicotinamide chromophores is evident. L-Malate binding, however, apparently produces only a perturbation of the adenine chromophore in such complexes. Since the coenzyme has been found to bind in an open conformation on the surface of the enzyme and the substrate analogs bind at or very near the dihydronicotinamide moiety binding site, protein conformational changes are implicated during ternary complex formation with D-malate which can effect the adenine chromophore at some distance from the substrate binding site.     

The interaction and inhibition of muscle lactate dehydrogenase by the alkaloid caffeine

Kinetic analysis showed that the alkaloid caffeine is a competitive inhibitor of the enzyme lactate dehydrogenase with respect to substrate pyruvate, and a non-competitive inhibitor with respect to the coenzyme NADH. The inhibitor constant Ki is 0,54 mM. Scatchard analysis determined the dissociation constant for a single independent binding site of the ternary lactate dehydrogenase - NADH - caffeine complex (KE-NADH-CAFFEINE) and the number of binding sites to be 0,14 mM and 3,83 respectively. Caffeine binds to a hydrophobic domain in the substrate binding site. Alternate nucleophilic - electrophilic functionalities within the inhibitor molecule are proposed to be the fundamental reason for the inhibition.     

Production of essential L-branched-chain amino acids in bioreactors containing artificial cells immobilized multienzyme systems and dextran-NAD+

We prepared artificial cells each containing leucine dehydrogenase (EC 1.4.1.9), urease (EC 3.5.1.5), soluble dextran-NAD(+), and one of the following coenzyme regenerating dehydrogenases: glucose dehydrogenase (EC 1.1.1.47); yeast alcohol dehydrogenase (EC 1.1.1.1); malate dehydrogenase (EC 1.1.1.37); or lactate dehydrogenase (EC 1.1.1.27). Artificial cells were packed in small columns. L-Leucine, L-valine, and L-isoleucine were continuously produced with simultaneous dextran-NADH regeneration. The maximum production ratios depended on the coenzyme regenerating systems used: 83-93% for D-glucose and glucose dehydrogenase system; 90% for ethanol and yeast alcohol dehydrogenase system; 45-55% for L-malate and malate dehydrogenase system; and 64-78% for L-lactate and lactate dehydrogenase system. Kinetic experiments were also carried out. The apparent K(m) values are as follows: 0.33 mM for alpha-ketoisocaproate (KIC); 0.51 mM for alpha-ketoisovalerate (KIV); 0.58 mM for DL-alpha-keto-beta-methyl-n-valerate (KMV); 3.52 mM for urea; 27.82 mM for D-glucose; 3.89 mM for ethanol; 3.02 mM for L-malate; and 16.67 mM for L-lactate. Kinetic analysis showed that KIC, KIV, and KMV were all competitive inhibitors in the reactions catalyzed by leucine dehydrogenase. Their inhibitor constants were the corresponding K(m) values.     

Comparison of lactate and malate dehydrogenases: Fluorescence and thermodynamic properties

Equilibrium, thermochemical, and time-resolved fluorescence measurements have been carried out in order to compare pig heart lactate dehydrogenase (LDH) and cytoplasmic malate dehydrogenase (MDH). The differences in the thermodynamic parameters for binding of NADH and NAD+ show the same pattern for both enzymes. The stronger binding of NADH is entropy-based, which can be understood as reflecting electrostatic interactions. The tryptophan fluorescence of MDH and LDH differ for the free enzymes and in quenching by NADH. The differences can be accounted for in terms of a single long-lived tryptophan residue present in LDH and not in MDH.     

Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase.

1. The pH-dependencies of the binding of NADH and reduced nicotinamide--benzimidazole dinucleotide to pig heart cytoplasmic malate dehydrogenase and lactate dehydrogenase are reported. 2. Two ionizing groups were observed in the binding of both reduced coenzymes to lactate dehydrogenase. One group, with pKa in the range 6.3--6.7, is the active-site histidine residue and its deprotonation weakens binding of reduced coenzyme 3-fold. Binding of both coenzymes is decreased to zero when a second group, of pKa 8.9, deprotonates. This group is not cysteine-165.3. Only one ionization is required to characterize the binding of the two reduced coenzymes to malate dehydrogenase. The group involved appears to be the active-site histidine residue, since its ethoxycarbonylation inhibits the enzyme and abolishes binding of reduced coenzyme. Binding of either reduced coenzyme increases the pKa of the group from 6.4 to 7.4, and deprotonation of the group is accompanied by a 10-fold weakening of coenzyme binding. 4. Two reactive histidine residues were detected per malate dehydrogenase dimer. 5. A mechanism which emphasizes the homology between the two enzymes is presented.     

Inhibition of lactate and malate dehydrogenase by dyes containing vinylsulfone and chlorotriazine groups

The difference spectra of lactate and malate dehydrogenase complexes with four native dyes containing vinylsulfonic and triazinic groups (light-resistant yellow 2KT, red-violet 2KT, etc.) were monitored in 0.1 M phosphate buffer pH 8.2 at 20 degrees C. The dissociation constants were calculated from the spectral data. The most stable complexes were lactate dehydrogenase--light-resistant yellow 2KT and malate dehydrogenase--light-resistant yellow 2KT ones. The values of delta H degree = 5.75 kcal/mole and standard thermodynamic parameters, delta G degree = -6.5 kcal/mole and delta S degree = 41.2 e. u., were calculated from the values of association constants for temperature dependence. The thermodynamic characteristics confirmed the key role of hydrophobic interactions in lactate dehydrogenase--reactive dye complex formation. All the dyes under study competitively inhibit lactate and malate oxidation by the corresponding dehydrogenases. The inhibition constants of both enzymes by the four dyes were determined at 20 degrees C in 0.1 M phosphate buffer pH 8.2. Light-resistant yellow 2KT appeared to be the most effective inhibitor of the enzymes.     

Studies on lactate dehydrogenase activity in the shrimp Palaemon serratus. Part 1: Characterization of the enzymes from the caudal muscle of Palaemon serratus

Palaemon serratus muscle lactate dehydrogenase (LDH) has been partially purified. The electrophoretic pattern of the LDH enzymes indicates that at least two molecular structures are present. The mean molecular weight is 130,000. The substrate and coenzyme dependence of the LDH system show non-Michaelian kinetics. This can be interpreted as being due to the presence of two binding sites in the enzyme which show negative effects. The behaviour of the two molecular species of LDH appears to be nearly identical in this respect. The study of the effect of temperature on the kinetic parameters of the LDH system shows the existence of a thermal dependence of K_m values. This may be related to developmental or environmental changes in the animal.     

Kinetic analysis of duck epsilon-crystallin with L-lactate dehydrogenase activity: determination of kinetic constants and comparison of substrate specificity.

A systematic analysis of the kinetic properties of duck lens epsilon-crystallin with lactate dehydrogenase [LDH, (E.C. 1.1.1.27)] activity was carried out by employing some 19 different alpha-keto acids as substrates for this NADH-dependent LDH-catalyzed reaction. The steady-state Michaelis and catalytic constants (Km, kcat) were determined for a broad range of organic compounds. The results provide important insights regarding the binding and affinity of substrates to active sites of this enzyme crystallin and indicate a great potential for the application of the stable epsilon-crystallin as a catalyst to the synthesis of some important chiral alpha-hydroxyacids in a convenient and efficient way. It is also demonstrated for the first time that in addition to the enzymatic activity of lactate dehydrogenase, duck epsilon-crystallin also possesses the enzymatic activity of malate dehydrogenase.     

The three-dimensional structure of porcine heart mitochondrial malate dehydrogenase at 3.0-A resolution

In a previous study, we reported the apparent similarity between a low resolution electron density map of mitochondrial malate dehydrogenase and a model of cytoplasmic malate dehydrogenase (Roderick, S. L., and Banaszak, L. J. (1983) J. Biol. Chem. 258, 11636-11642). We have since determined the polypeptide chain conformation and coenzyme binding site of crystalline porcine heart mitochondrial malate dehydrogenase by x-ray diffraction methods. The crystals from which the diffraction data was obtained contain four subunits of the enzyme arranged as a "dimer of dimers," resulting in a crystalline tetramer which possesses 222 molecular symmetry. The overall polypeptide chain conformation of the enzyme, the location of the coenzyme binding site, and the preliminary location of several catalytically important residues have confirmed the structural similarity of mitochondrial malate dehydrogenase to cytoplasmic malate dehydrogenase and lactate dehydrogenase.     

Glutamate-malate metabolism in liver mitochondria. A model constructed on the basis of mitochondrial levels of enzymes, specificity, dissociation constants, and stoichiometry of hetero-enzyme complexes.

The level of aspartate aminotransferase in liver mitochondria was found to be approximately 140 microM, or 2-3 orders of magnitude higher than its dissociation constant in complexes with the inner mitochondrial membrane and the high molecular weight enzymes (M(r) = 1.6 x 10(5) to 2.7 x 10(6)) carbamyl-phosphate synthase I, glutamate dehydrogenase, and the alpha-ketoglutarate dehydrogenase complex. The total concentration of aminotransferase-binding sites on these structures in liver mitochondria was more than sufficient to accommodate all of the aminotransferase. Therefore, in liver mitochondria, the aminotransferase could be associated with the inner mitochondrial membrane and/or these high molecular weight enzymes. The aminotransferase in these hetero-enzyme complexes could be supplied with oxalacetate because binding of aminotransferase to the high molecular weight enzymes can enhance binding of malate dehydrogenase, and binding of both malate dehydrogenase and the aminotransferase facilitated binding of fumarase. The level of malate dehydrogenase was found to be so high (140 microM) in liver mitochondria, compared with that of citrate synthase (25 microM) and the pyruvate dehydrogenase complex (0.3 microM), that there would also be a sufficient supply of oxalacetate to citrate synthase-pyruvate dehydrogenase.     

Enzymes of de novo pyrimidine biosynthesis in Babesia rodhaini

The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoa Babesia rodhaini has been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2); dihydroorotase (DHOase: E.C. 3.5.2.3); dihydroorotate dehydrogenase (DHO-DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine-5'-phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO-DHase, OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N-(phosphonacetyl)-L-aspartate was shown to be an inhibitor of the ATCase with an apparent Ki of 7 microM. Dihydro-5-azaorotate inhibited the DHO-DHase (Ki, 16 microM) and 5-azaorotate (Ki, 21 microM) was an inhibitor of the OPRTase. The UMP analog, 6-aza-UMP (Ki, 0.3 microM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product, UMP (Ki, 120 microM) and the purine nucleotide, XMP (Ki, 95 microM). Additionally, menoctone, a ubiquinone analog, was shown to inhibit DHO-DHase.     

Functional interrelations between isozymes of dehydrogenases in the intact and denervated rabbit muscles]

A correlation is shown to exist between malate dehydrogenase (MDH), lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (glycerol-3-PDH activity values, lactate/pyruvate and malate/oxaloacetate coefficients, MDH and LDH isozyme spectra and kinetic properties of LDH isozymes in soluble fractions of cytoplasm from intact rabbit m. soleus (red), m. gastrocnemius (mixed) and m. quadratus lumborum (white). In denervated soleus and gastrocnemius the cytoplasmic MDH/LDH, mitochondrial MDH/LDH, MDH mitochondrial/MDH cytoplasmic activity ratios, concentrations of substrates and isozyme spectra of MDH and LDH tend to equalize. The obtained results indicate the importance of isozyme composition and total activity ratios of the dehydrogenases for regulation of pyruvate and NADH metabolic pathways.     

Inhibition of Lactate Production in Rat Brain Extracts and Synaptosomes by 3-[4-(Reduced 3-Pyridine Aldehyde-Adenine Dinucleotide)]-Pyruvate

In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.     

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.     

Sodium dodecyl sulfate, concurrent inhibitor of several dehydrogenases

The effect of sodium dodecyl sulfate on the activity of highly purified or crystalline enzymes has been studied. The enzymes were: lactate dehydrogenase (LDH), malate dehydrogenase (MDH). isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6P-DH), lipase, alkaline phosphatase. Sodium dodecyl sulfate, always under the critical micellar concentration, shows a selective inhibitory effect. A kinetic analysis of the inhibitory action on LDH, MDH, ICDH and G6P-DH was also carried out.     

Lactate and malate dehydrogenase binding to the microsomal fraction from chicken liver

Chicken liver microsomal fractions show lactate and malate dehydrogenase activities which behave differently with respect to successive extractions by sonication in 0.15 M NaCl, 0.2% Triton X-100 and 0.15 M NaCl, respectively. The Triton X-100-treated pellet did not show malate dehydrogenase activity but exhibited a 10-fold increase in lactate dehydrogenase activity with respect to the sonicated pellet. Total extracted lactate and malate dehydrogenase activities were, respectively, 7.5 and 1.7 times higher than that in the initial pellet. Different isoenzyme compositions were observed for cytosoluble and microsomal extracted lactate and malate dehydrogenases. When the ionic strength (0-500 mM) or the pH values (6.1-8.7) of the media were increased, an efficient release of lactate dehydrogenase was found at NaCl 30-70 mM and pH 6.6-7.3. Malate dehydrogenase solubilization under the same conditions was very small, even at NaCl 500 mM, but it attained a maximum in the 7.3-8.7 pH range. Cytosoluble lactate dehydrogenase bound in vitro to 0.15 M NaCl-treated (M2) and sonicated (M3) microsomal fractions but not to the crude microsomal fraction (M1). Particle saturation by lactate dehydrogenase occurred with M2 and M3, which contained binding sites with different affinities. Cytosoluble malate dehydrogenase did not bind to M1, M2 and M3 fractions, however, a little binding was found when purified basic malate dehydrogenase was incubated with M2 or M3 fractions.     

Alteration of coenzyme specificity of lactate dehydrogenase from Thermus thermophilus by introducing the loop region of NADP(H)-dependent malate dehydrogenase

Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k cat/Km(coenzyme) was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.     

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.