Effects of quinones on NADH-dependent enzymatic bioluminescent systems

The effects of a number of quinones on the bioluminescence characteristics of a three-component enzymatic system containing alcohol dehydrogenase, bacterial luciferase, and NADH-FMN oxidoreductase were studied to find the most sensitive kinetic parameters of the system intended to be used in biological testing. Both direct and back reactions catalyzed by alcohol dehydrogenase were studied in the presence and in the absence of quinones. The kinetic parameters of the bioluminescent system were found to depend on the redox potentials and concentrations of quinones. The quinone-induced effects were shown to be associated with changes in the NAD⁺/NADH ratio in the chain of NADH-dependent enzymes. The three-enzyme system based on alcohol dehydrogenase is suggested as a bioluminescence test for ecological monitoring of waste water.     

Redox compounds influence on the NAD(P)H:FMN-oxidoreductase-luciferase bioluminescent system.

A review of the mechanisms of the exogenous redox compounds influence on the bacterial coupled enzyme system: NAD(P)H:FMN-oxidoreductase-luciferase has been done. A series of quinones has been used as model organic oxidants. The three mechanisms of the quinones' effects on bioluminescence were suggested: (1) inhibition of the NADH-dependent redox reactions; (2) interactions between the compounds and the enzymes of the coupled enzyme system; and (3) intermolecular energy migration. The correlation between the kinetic parameters of bioluminescence and the standard redox potential of the quinones proved that the inhibition of redox reactions was the key mechanism by which the quinones decrease the light emission intensity. The changes in the fluorescence anisotropy decay of the endogenous flavin of the enzyme preparations showed the direct interaction between quinones and enzymes. It has been demonstrated that the intermolecular energy migration mechanism played a minor role in the effect of quinones on the bioluminescence. A comparative analysis of the effect of quinones, phenols and inorganic redox compounds on bioluminescent coupled enzyme systems has been carried out.     

Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase.

Reductive methylation of lysine residues activates liver alcohol dehydrogenase in the oxidation of primary alcohols, but decreases the activity of the enzyme towards secondary alcohols. The modification also desensitizes the dehydrogenase to substrate inhibition at high alcohol concentrations. Steady-state kinetic studies of methylated liver alcohol dehydrogenase over a wide range of alcohol concentrations suggest that alcohol oxidation proceeds via a random addition of coenzyme and substrate with a pathway for the formation of the productive enzyme-NADH-alcohol complex. To facilitate the analyses of the effects of methylation on liver alcohol dehydrogenase and factors affecting them, new operational kinetic parameters to describe the results at high substrate concentration were introduced. The changes in the dehydrogenase activity on alkylation were found to be associated with changes in the maximum velocities that are affected by the hydrophobicity of alkyl groups introduced at lysine residues. The desensitization of alkylated liver alcohol dehydrogenase to substrate inhibition is identified with a decrease in inhibitory Michaelis constants for alcohols and this is favoured by the steric effects of substituents at the lysine residues.     

Ethanol-metabolizing enzymes from human testis

Testicular ethanol-metabolizing enzymes (alcohol dehydrogenase, microsomal ethanol-oxidizing system, catalase) were investigated. Alcohol dehydrogenase was purified to homogeneity and its main kinetic parameters were analyzed. It was shown that alcohol dehydrogenase corresponds to class III isozymes and does not participate in ethanol oxidation. The testicular microsomal ethanol-oxidizing activity does not exceed 0.02 nmol/min/mg of protein. The activity of catalase and its peroxidase component is far lower in the testes than in the liver. On the whole, testicular tissue is rather inactive in respect of ethanol oxidation.     

Effect of pH on the process of ternary-complex interconversion in the liver-alcohol-dehydrogenase reaction.

1. Kinetic relationships referring to multiple-turnover conditions have been derived for the slowest exponential transient appearing in two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism. The validity of these and previously derived theoretical relationships for this mechanism has been tested by application to the liver alcohol dehydrogenase reaction. 2. All essential features of the transient-state kinetics of alcohol oxidation by NAD+ in the liver alcohol dehydrogenase system can be qualitatively and quantitatively explained in view of the compulsory-order mechanism in the proposed scheme. There is no kinetic evidence for any half-of-the-sites reactivity of the enzyme. A consistent set of rate constants is reported for the enzymic oxidation of benzyl alcohol at pH 8.75. 3. Transient-state rate parameters for benzyl alcohol/benzaldehyde catalysis by liver alcohol dehydrogenase have been determined at different pH. The interpretation of such rate parameters is critically discussed with reference to their informative value for the purpose of determination of rate constants (k and k') for the process of ternary-complex interconversion in the proposed scheme. It is concluded that the apparent rate constant (k') for hydride transfer from benzyl alcohol to NAD+ is dependent on a proton dissociation step with a pKa of 6.4, whereas the rate constant (k) for hydride transfer from NADH to benzaldehyde exhibits no corresponding dependence on proton association. 4. The asymmetric pH dependence of the forward and reverse rate of ternary-complex interconversion during liver alcohol dehydrogenase catalysis appears to reflect an obligatory step of alcohol/alcoholate ion equilibration occurring at the ternary-complex level. It is suggested that the observed pKa 6.4 dependence of the transient rate of alcohol oxidation can be attributed to a coupled acid-base system involving minimally the enzyme-bound alcohol and the protein residues Ser-48 and His-51.     

Kinetic studies on long-chain alcohol turnover by yeast alcohol dehydrogenase within the transition from real solution to emulsion or suspension]

The enzymatic oxidation of hexanol, decanol, and tetradecanol by yeast alcohol dehydrogenase was studied. The enzyme was found to catalyze not only conversion in the real aqueous solution of the substrates, but also at the surface of undissolved substrate particles. The kinetic parameters varied on transition from the real solution to dispersion, in dependence on the chain length of the substrate.     

An enzymatic model for rat liver alcohol dehydrogenase in the presence of low-Km aldehyde dehydrogenase

Four isoenzymes of aldehyde dehydrogenase were partially purified from rat liver mitochondria by hydroxylapatite chromatography and gel filtration. While three forms display low affinity for acetaldehyde, the fourth is active at extremely low aldehyde concentrations (Km less than or equal to 2 microM) and allows the oxidation of the acetaldehyde formed by catalysis of alcohol dehydrogenase at pH 7.4. Different models of alcohol dehydrogenase have been examined by analysis of progress curves of ethanol oxidation obtained in the presence of low-km aldehyde dehydrogenase. According to the only acceptable model, when the acetaldehyde concentration is kept low by the action of aldehyde dehydrogenase, NADH no longer binds to alcohol dehydrogenase, but acetaldehyde still competes with ethanol for the active site of the enzyme. The seven kinetic parameters of the two enzymes (four for alcohol dehydrogenase and three for aldehyde dehydrogenase) and the equilibrium constant of the reaction catalyzed by alcohol dehydrogenase have been determined by applying a new fitting procedure here described.     

Evaluation of simple enzyme kinetics by response surface modelling

The activity of a horse liver alcohol dehydrogenase catalysed reduction of cyclohexanone was investigated by using a central composite circumscribed design in which two parameters (pH and cyclohexanone concentration) were varied. By log transformation of the substrate concentration an adequate model could be obtained from which reliable kinetic constants and pH profiles were determined.     

Affinity chromatography of lactate dehydrogenase on immobilized nucleotides

The interaction of two isoenzymes of lactate dehydrogenase from pig heart muscle (H(4)) and rabbit skeletal muscle (M(4)), with immobilized nucleotides was examined: the effects of pH and temperature on the binding of lactate dehydrogenase were studied with immobilized NAD(+) matrices. The influence of substrate, product and sulphite on the binding of heart muscle lactate dehydrogenase to immobilized NAD(+) was investigated. The interaction of both lactate dehydrogenase isoenzymes with immobilized pyridine and adenine nucleotides and their derivatives were measured. The effects of these parameters on the interaction of lactate dehydrogenase with immobilized nucleotides were correlated with the known kinetic and molecular properties of the enzymes in free solution.     

Use of a polymer-bound flavin derivative for the rapid regeneration of NAD(P)+ from NAD(P)H in dehydrogenase systems

An aldehyde derivative of riboflavin was covalently attached by reductive alkylation to soluble polycationic supports. The flavopolymers so obtained were stable under operational conditions. The catalytic efficiency towards oxidation of NADH by these flavopolymers was demonstrated, and the kinetic parameters (Km and kcat) revealed an overall catalytic efficiency (kcat/Km) 185-fold greater compared to riboflavin. Various factors affecting the chemical regeneration of NAD+ from NADH such as pH, ionic strength, nature of the buffer etc. were studied. The most interesting result was the highly favourable influence of borate ions which increased the reaction rate by a factor 2-4 compared to the other buffers. The flavopolymers are very effective for in situ recycling of NAD(P)+. With up to 300-fold NADH----NAD+ conversions for the system using yeast alcohol dehydrogenase and up to 1500-fold NADPH----NADP+ regenerations for the system using glucose-6-phosphate dehydrogenase. These flavopolymers are superior to previous chemical recycling systems.     

Dehydrogenase-dependent ethanol metabolism in deer mice (Peromyscus maniculatus) lacking cytosolic alcohol dehydrogenase. Reversibility and isotope effects in vivo and in subcellular fractions

Elimination of [2H]ethanol in vivo as studied by gas chromatography/mass spectrometry occurred at about half the rate in deer mice reported to lack alcohol dehydrogenase (ADH-) compared with ADH+ deer mice and exhibited kinetic isotope effects on Vmax and Km (D(V/K] of 2.2 +/- 0.1 and 3.2 +/- 0.8 in the two strains, respectively. To an equal extent in both strains, ethanol elimination was accompanied by an ethanol-acetaldehyde exchange with an intermolecular transfer of hydrogen atoms, indicating the occurrence of dehydrogenase activity. This exchange was also observed in perfused deer mouse livers. Based on calculations it was estimated that at least 50% of ethanol elimination in ADH- deer mice was caused by the action of dehydrogenase systems. NADPH-supported cytochrome P-450-dependent ethanol oxidation in liver microsomes from ADH+ and ADH- deer mice was not stereoselective and occurred with a D(V/K) of 3.6. The D(V/K) value of catalase-dependent oxidation was 1.8, whereas a kinetic isotope effect of cytosolic ADH in the ADH+ strain was 3.2. Mitochondria from both ADH+ and ADH- deer mice catalyzed NAD+-dependent ethanol oxidation and NADH-dependent acetaldehyde reduction. The kinetic isotope effects of NAD+-dependent ethanol oxidation in the mitochondrial fraction from ADH+ and ADH- deer mice were 2.0 +/- 0.1 and 2.3 +/- 0.3, respectively. The results indicate only a minor contribution by cytochrome P-450 to ethanol elimination, whereas the isotope effects are consistent with ethanol oxidation by the catalase-H2O2 system in ADH- deer mice in addition to the dehydrogenase systems.     

Enhanced stability of alcohol dehydrogenase by non-covalent interaction with polysaccharides

Non-covalent interaction of alcohol dehydrogenase with polysaccharides was studied using three neutral and three anionic polysaccharides. The process of interaction of alcohol dehydrogenase with gum Arabic was optimized with respect to the ratio of enzyme to gum Arabic, pH, and molarity of buffer. Alcohol dehydrogenase–gum Arabic complex formed under optimized conditions showed 93 % retention of original activity with enhanced thermal and pH stability. Lower inactivation rate constant of alcohol dehydrogenase–gum Arabic complex within the temperature range of 45 to 60 °C implied its better stability. Half-life of alcohol dehydrogenase–gum Arabic complex was higher than that of free alcohol dehydrogenase. A slight increment was observed in kinetic constants (K _m and V _max) of gum Arabic-complexed alcohol dehydrogenase which may be due to interference by gum Arabic for the binding of substrate to the enzyme. Helix to turn conversion was observed in complexed alcohol dehydrogenase as compared to free alcohol dehydrogenase which may be responsible for observed stability enhancement.     

Inactivation of alcohol dehydrogenase in rice seedlings is related to a microsomal fatty acid alpha-oxidation system

The selective inactivation of alcohol dehydrogenase by the inactivator found in the microsomal fraction of rice (Oryza sativa) seedlings growing in air (Shimomura, S. & Beevers, H. (1983) Plant Physiol. 71, 736-741; 742-746) was further studied. This inactivation was found to be essentially dependent on the presence of free fatty acids. The specificity for fatty acids and the inhibitory effects of imidazole, 2-hydroxyfatty acids and dithiothreitol on the inactivation were all consistent with the properties of the fatty acid alpha-oxidation system in plants. Both O2 consumption and decarboxylation of fatty acid due to alpha-oxidation were also demonstrated in rice microsomes. When purified rice alcohol dehydrogenase was added to the alpha-oxidation system in rice microsomes, the decarboxylation of fatty acid was inhibited, and the cysteinyl residues of alcohol dehydrogenase were oxidized. The oxidation of two cysteinyl residues per monomer resulted in the complete inactivation of the enzyme. The activity of the inactivator in the isolated microsomes was gradually lost during storage and was rapidly lost upon heating. The inactivation of alcohol dehydrogenase was observed even when the enzyme was separated from microsomes by a dialysis membrane. These results indicate that the inactivation of alcohol dehydrogenase is closely related to fatty acid alpha-oxidation. We postulate that an intermediate of alpha-oxidation is the inactivator.     

De novo design and utilization of photolabile caged substrates as probes of hydrogen tunneling with horse liver alcohol dehydrogenase at sub-zero temperatures: a cautionary note

In order to understand the influence of protein dynamics on enzyme catalysis and hydrogen tunneling, the horse liver alcohol dehydrogenase (HLADH) catalyzed oxidation of benzyl alcohol was studied at sub-zero temperatures. Previous work showed that wild type HLADH has significant kinetic complexity down to -50 degrees C due to slow binding and loss of substrate [S.-C. Tsai, J.P. Klinman, Biochemistry, 40 (2001) 2303]. A strategy was therefore undertaken to reduce kinetic complexity at sub-zero temperatures, using a photolabile (caged) benzyl alcohol that prebinds to the enzyme and yields the active substrate upon photolysis. By computer modeling, a series of caged alcohols were designed de novo, synthesized, and characterized with regard to photolysis and binding properties. The o-nitrobenzyl ether 15, with a unique long tail, was found to be most ideal. At sub-zero temperatures in 50% MeOH, a two-phase kinetic trace and a rate enhancement by the use of 15 were observed. Despite the elimination of substrate binding as a rate-limiting step, the use of caged benzyl alcohol does not produce a measurable H/D kinetic isotope effect. Unexpectedly, the observed fast phase corresponds to multiple enzyme turnovers, based on the stoichiometry of the substrate to enzyme. Possible side reactions and their effects, such as the re-oxidation of bound NADH and the dissipation of photo-excitation energy, may offer an explanation for the observed multiple-turnovers. The lack of observable deuterium isotope effects offers a cautionary note for the application of caged substrates to isolate and study chemical steps of enzyme reactions, particularly when NADH is involved in the reaction pathway.     

Specific interaction among some enzymes and sodium dodecyl sulfate

The effect of 1-butanesulfonic acid sodium salt and sodium dodecyl sulfate on the activity of highly purified and crystalline enzymes with marked differences in structure and function has been studied. The enzymes were: alcohol dehydrogenase; lactate dehydrogenase; malate dehydrogenase; isocitrate dehydrogenase; glucose-6-phosphate dehydrogenase; lipase; alkaline phosphatase. While 1-butanesulfonic acid sodium salt, at the studied concentrations, resulted generally inactive, sodium dedecyl sulfate showed a selective inhibitory effect, always under the critical micellar concentration. A kinetic analysis of the inhibitory action was also carried out.     

Substituent effects in alkylated liver alcohol dehydrogenases

Alcohol dehydrogenase from horse liver was reductively alkylated with aldehydes having varied alkyl substituents. Kinetic studies of alkylated liver alcohol dehydrogenases which were modified in the absence and in the presence of NADH indicate that the alkylation of the specific lysine residues generally activates the enzyme by increasing Michaelis and inhibition constants for substrates and maximum velocities for the reactions. These kinetic parameters were analyzed in terms of electronic, steric, and hydrophobic effects of alkyl substituents. The hydrophilic character of the lysine residues is the most important factor which affects all kinetic parameters, particularly K_ia and V₂. In addition, the nucleophilic character of the lysine residues enhances the enzyme activity by increasing the maximum velocity of ethanol oxidation and the affinity of alcohol dehydrogenase for NADH and acetaldehyde. The steric interaction at the lysine residues favors the affinity of the enzyme for NADH and ethanol.     

Comparison of two-phase lipase-catalyzed esterification on micro and bench scale

Lipase type B from Candida antarctica was used to catalyze the esterification of propionic acid and 1-butanol in a water/n-decane two-phase system on micro and on bench scale. The reaction was described by a Ping Pong Bi Bi mechanism with alcohol inhibition. The kinetic parameters on micro and bench scale were compared; no significant differences were found. Furthermore, effects of temperature on activation and inactivation of the enzyme were found to be similar on micro and bench scale. Therefore, parameters found on either scale can be used for the other scale. Enzyme kinetic parameters can be determined on a micro scale, with very low consumption of reagents and catalyst, and then be applied to bench scale. This can reduce the cost of optimizing enzyme processes by downscaling.     

Calculating molar absorptivities for quinones: application to the measurement of tyrosinase activity

The molar absorptivities of the quinones produced from different o-diphenols, triphenols, and flavonoids were calculated by generating the respective quinones through oxidation with an excess of periodate. Oxidation of these substrates by this reagent was analogous to oxidation by tyrosinase with molecular oxygen, although the procedure showed several advantages over the enzymatic method in that oxidation took place almost immediately and quinone stability was favored because no substrate remained. The o-diphenols studied were pyrocatechol, 4-methylcatechol, 4-tert-butylcatechol, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylethylamine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, and caffeic acid; the triphenols studied were pyrogallol, 1,2,4-benzenetriol, 6-hydroxydopa, and 6-hydroxydopamine; and the flavonoids studied were (+)catechin, (-)epicatechin, and quercetin. In addition, the stability of the quinones generated by oxidation of the compounds by [periodate]0/[substrate]0 < 1 was studied. Taking the findings into account, tyrosinase could be measured by following o-quinone formation in rapid kinetic studies using the stopped-flow method. However, measuring o-quinone formation could not be useful for steady-state studies. Therefore, several methods for following tyrosinase activity are proposed, and a kinetic characterization of the enzyme's action on these substrates is made.     

ENZYMES CATALYSING ETHANOL METABOLISM IN NEURAL AND SOMATIC TISSUES OF THE RAT

Abstract— The enzymes catalysing ethanol metabolism, alcohol dehydrogenase (EC 1.l.1.1) and aldehyde dehydrogenase (EC 1.2.1.3), were assayed in a variety of neural and somatic tissues of the rat, the human counterparts of which are known to be vulnerable to excessive ethanol. The activity of alcohol dehydrogenase was assayed by the coupled oxidation of ethanol and reduction of lactaldehyde, a method which we have recently found to be sufficiently sensitive and specific to measure the relatively low levels of activity in whole brain. Detectable activities of these enzymes were found in peripheral nerve, skeletal muscle, retina, optic nerve and various regions of brain, as well as in a variety of non-neural tissues. The levels of the enzymic activities in all tissues were markedly lower than those of liver, but probably sufficient to perform a local function in the metabolism of ethanol or other endogenous substrates. The activity of alcohol dehydrogenase in the various tissues, like that of liver, was confined to the cytosol and exhibited kinetic properties and responses to inhibitors almost identical to those of the liver enzyme. We consider the results to be consistent with the hypothesis that the pathological effects of alcohol may be related, at least in part, to local mechanisms for the metabolism of alcohol.     

Effect of quinones and phenols on the triple‐enzyme bioluminescent system with protease

The study addressed the effects of redox‐active compounds on trypsin activity. Series of organic oxidizers (quinones) and reducers (phenols) were chosen as model redox‐active compounds. Trypsin activity was quanti?ed by bioluminescent technique. Interactions of these compounds with trypsin were studied by ?uorescent and light absorption methods. Luminescence intensity decay constants in the reduced nicotinamidadeninedinucleotide (NADH): ?avinmononucleotide (FMN)‐oxidoreductase (R)–luciferase (L)–trypsin (T) (R + L + T) triple‐enzyme system were calculated and compared in the presence of different concentrations of quinones and phenols. The triple‐enzyme system was shown to be sensitive to quinones and not sensitive to phenols. It has been found that the effects produced by quinones on the coupled enzyme system (R + L) and on the trypsin molecule (T) are not related. The conclusions were extrapolated to the properties of other proteases and antiproteases. Copyright © 2003 John Wiley & Sons, Ltd.