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The nucleotides DSI and DSII induced during a slowdown in growth of E. coli have been characterized using chemical and biochemical analysis and by enzymic and alkaline fragmentation. DSI consists a coenzyme A and glutathione joined by a disulfide linkage. DSI could be isolated either containing Fe(III) with an A250:260 ratio of 1.05 or not containing iron with an A250:260 of 0.87. DSII (isolated in 10% the yield of DSI) is a coenzyme A disulfide dimer that also contains two molecules of glutamic acid. DSI was a substrate for NADPH-dependent CoAS-SG reductase (EC 1.6.4.6) which was present in crude extracts of E. coli. The specific activity of CoAS-SG reductase increased during growth from early log phase into stationary phase and during a shift from aerobic to anaerobic growth.
Dormant spores of Bacillus megaterium were found to contain approximately 850 pmol of coenzyme A (CoA) per milligram of dry weight. Of this total, less than 1.5% was acetyl-CoA, 25% was CoA-disulfide, 43% was in disulfide linkage to protein, and the remainder was the free thiol. Dormand spores of Bacillus cereus and Clostridium bifermentans contained 700 and 600 pmol of CoA per milligram of dry weight, respectively; in both species approximately 45% of the CoA 45% of the CoA was in disulfide linkage to protein. During germination of spores of all three species, greater than 75% of the CoA-protein disulfides were cleaved. In B. megaterium, cleavage of these disulfides during spore germination did not require exogenous metabolites and occurred at about the same time as the initiation of germination. Much of the CoA was converted to acetyl-CoA at this time. Dormant spores also contained reduced nicotinamide adenine dinucleotide-dependent CoA-disulfide reductase at levels higher than those in other stages of growth. The level of total CoA in the growing cells was two- to three-fold higher than in spores. This level remained constant throughout growth and sporulation, but less than 2% of the total cellular CoA was in disulfide linkage to protein until late in sporulation. The CoA-protein disulfides accumulated exclusively within the developing spore at about the time when dipicolinic acid was accumulated.
The binding rate of pyridoxal '5-phosphate (Pxa-P) to apotryptophanase and the dissociation rate of the coenzyme from holotryptophanase were able to be determined by following the enzyme activity in continuous flow reactions on a column of immobilized tryptophanase. When the enzyme activity was assayed continuously in the flow system in the absence of coenzyme added to the reaction mixture, immobilized holotryptophanase lost gradually its initial activity owing to dissociation of coenzyme. The coenzyme dissociation at a given concentration of substrate (tryptophan) followed first-order kineticsmin a low substrate concentration range below the Km value, a more decreased rate constant was obtained for the coenzyme dissociation. This indicates that the coenzyme is more dissociable from the apoenzyme-coenzyme-substrate complex (ECS complex) rather than from the apoenzyme-coenzymecomplex (holoenzyme). Immobilized tryptophanase freed of coenzyme restored rapidly its original activity, when the assay mixture containing a given concentration of substrate and Pxa-P was passed through the immobilized enzyme column. The coenzyme binding at a given coenzyme concentration followed first-order kinetics, but the rate was not first order in regard to the coenzyme concentration. A plot of the reciprocal of the first-order rate constant obtained vs. the reciprocal of the coenzyme binding occurs in a two-step fashion; the first step is rapid and the second step is rate determining. Both the dissociation constant for the first step and the rate constant for the second step were shown to be independent of the substrate concentration. This means that Schiff base formation between Pxa-P and tryptophan in the assay mixture has no effect on the binding of Pxa-P to apoenzyme. The coenzyme dissociation constant at a given substrate concentration was calculated from both the rate constant of the coenzyme binding and the rate constant of the coenzyme dissociation. The values obtained by this method at different substrate concentrations were almost identical with those measured at the corresponding substrate concentrations directly by an ordinary method.
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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.
Adriamycin inhibits the succinoxidase system and the NADH-oxidase system. Both of the intact mitocondrial enzymes and the pentane-extracted preparations are inhibited. The inhibition can be prevented by a molar ratio of coenzyme to adriamycin of 3:1 for coenzyme Q10 (ubiquinone), 5:1 for coenzyme Q7, and 5:1 for coenzyme Q4. Prevention of inhibition was observed in the decreasing order of coenzyme Q10 greater than coenzyme Q7 greater than H6 coenzyme Q4 greater than coenzyme Q4. Adriamycinone was three times more inhibitory than adriamycin, which is compatible with a less polar fragment necessary to inhibit the lipoidal coenzyme Q10. Daunomycinone was not inhibitory at a concentration at which adriamycinone is effective, indicating that the hydroxyl group of the latter could be binding at the receptor, since it should not influence electron transfer or rings B and C.
Effect of BCG, coenzyme Q10, or their combination on ATPase activity in spleen lymphocytes of tumor-bearing rats was investigated in relation to changes in the content of individual coenzyme Q homologs in these cells. Contents of both coenzyme Q9 and Q10 in spleen lymphocytes significantly decreased in the late stage of Donryu rats bearing Sato lung carcinoma. Oligomycin-sensitive ATPase activity in spleen lymphocytes was also significantly depressed in this stage. The depressed, oligomycin-sensitive ATPase activity was significantly recovered by a 3-time intramuscular administration of coenzyme Q10 emulsified with ethanol and saline, and the decreased contents of coenzymes Q9 and Q10 were slightly restored by this treatment. This enzyme activity was also significantly recovered by an intravenous administration of BCG, and was elevated more by the combined treatment with BCG and the emulsified coenzyme Q10. These results suggest that the combined treatment with BCG and emulsified coenzyme Q10 can contribute to the improvement of the depressed bioenergetics in lymphocytes of tumor-bearing animals, and that this combined effect of BCG and emulsified coenzyme Q10 might be based on the combination of their individual activating effect on lymphocytes.
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The administration of mevalonic acid to rats by intravenous injection resulted in a dose- and time-dependent increase in the activity of cholesterol 7alpha-hydroxylase in the liver microsomal fraction, a decrease in the microsomal activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and no significant change in the activity of acyl-coenzyme A:cholesterol acyltransferase or in the concentration of free and of esterified cholesterol in the liver microsomal fraction. However, the increased hepatic cholesterogenesis that follows the injection of mevalonic acid resulted in an increase of the size of the intracellular pool of cholesterol that is in the environment of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acts as substrate for cholesterol 7alpha-hydroxylase. The administration of mevalonic acid to rats by stomach tube resulted in an increase in the activity of cholesterol 7alpha-hydroxylase and of acyl-coenzyme A:cholesterol acyltransferase and in the concentration of cholesterol esters in the liver microsomal fraction, while there was a considerable decrease in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
A number of 2-(methylthio)ethanesulfonate (methyl-coenzyme M) analogues were synthesized and investigated as substrates for methyl-coenzyme M reductase, an enzyme system found in extracts of Methanobacterterium thermoautotrophicum. Replacement of the methyl moiety by an ethyl group yielded an analogue which served as a precursor for ethane formation. Propyl-coenzyme M, however, was not converted to propane. Analogues which contained additional methylene carbons such as 3-(methylthio)propanesulfonate or 4-(methylthio)butanesulfonate or analogues modified at the sulfide or sulfonate position, N-methyltaurine and 2-(methylthio)ethanol, were inactive. These analogues, in addition to a number of commercially available compounds, also were tested for their ability to inhibit the reduction of methyl-coenzyme M to methane. Bromoethanesulfonate and chloroethanesulfonate proved to be potent inhibitors of the reductase, resulting in 50% inhibition at 7.9 X 10(6) M and 7.5 X 10(5) M. Analogues to coenzyme M which contained modifications to other regions were evaluated also and found to be weak inhibitors of methane biosynthesis.
The reduction of cytochrome c by the reduced form of the 6-decyl analogue of coenzyme Q follows first-order kinetics with respect to cytochrome c and increases in a linear manner with added mitochondrial protein. The activity is completely sensitive to antimycin A in whole cell extracts of yeast as well as in isolated mitochondria and fractionates with markers for the mitochondrial electron-transport chain. The presence of both cytochrome b and c1 in an approximately 2:1 ratio appears essential for enzymatic activity. Reduced coenzyme Q-cytochrome c reductase obeys Michaelis-Menten kinetics when assayed in mitochondria obtained from a yeast strain lacking coenzyme Q. Both reduced nitotinamide adenine dinucleotide and succinate:cytochrome c reductase activities were not detectable in six coenzyme Q deficient strains tested, but were restored after addition of the oxidized form of the coenzyme Q analogue. No marked difference in the concentration of the analogue required to restore the two activities was observed.
A sensitive micromethod for the determination of Coenzyme A and its esters down to about 0.2 pmol in a volume of 10 microliters and of the activity of citrate synthase is outlined. Epidermal material from healthy and psoriatic skin was utilized in microgram quantity as tissue source. The assay utilizes the ketoglutarate dehydrogenase reaction to yield NADH on addition of free Coenzyme A and the subsequent measurement of NADH by a bioluminescent reaction with Acromobacter fischerii. The total Coenzyme A content in six healthy subjects measured in stratum Malpighii was 1.58 +/- 0.19 mmol per kg dry weight. In six psoriatic patients non-involved and involved epidermis contained 1.51 +/- 0.27 and 1.50 +/- 0.25 mmol/kg, respectively. Long-chain acyl-Coenzyme A comprised about 20% in lesion-free skin and 60% of total content in the involved psoriatic epidermis. The activity of citrate synthase in basal layers of healthy epidermis was 0.30 +/- 0.04 mkat/kg dry weight.
S-Trifluoroacetonyl-coenzyme A has been synthesized in 87% yield by reaction of 1,1,1-trifluoro-3-bromopropanone with trilithium coenzyme A in presence of pyridine. The compound was characterized by its ultraviolet absorption spectrum and 1H and 19F nuclear magnetic resonance spectra. The alpha-methylene protons of the S-trifluoroacetonyl group exchanged with D2O and showed a pKa of 9.85 in S-trifluoroacetonylmercaptoethanol. S-Trifluoroacetonyl-coenzyme A is a competitive inhibitor of porcine heart citrate synthetase (Ki = 0.16 mM). It forms a binary complex with the enzyme and a ternary complex with enzyme/oxaloaetate binary complex, as evidenced ty the 19F shift. S-Trifluoracetonyl-coenzyme A and S-trifluoroacetonylmercaptoethanol form weak to moderately strong complexes with alpha-cyclodextrin and show little or no interaction with the methylglucose polysaccharide and lipopolysaccharides from Mycobacterium smegmatis [Smith, W. L., & Ballou, C. E. (1973) J. Biol. Chem. 248, 7118]. S-Trifluoroacetonylmercaptoethanol probably forms an inclusion complex with alpha-cyclodextrin because the interaction is reversed by compounds that do form inclusion complexes.
1. The NAD analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-NAD, was covalently bound to horse liver alcohol dehydrogenase in a carbodiimide-mediated reaction and in such a way that it was active with the very same enzyme molecule to which it was coupled. 2. The degree of substitution, i.e. the number of NAD analogues per enzyme subunit, could be varied (0.3-1.6). In one preparation 1.6 coenzyme molecules were bound per subunit; the alcohol dehydrogenase activity of this preparation was 40% of the activity obtained after addition of free NAD in excess. 3. It was calculated that every fourth active site of this preparation was provided with a covalently bound functioning coenzyme analogue, and that this analogue had a cycling rate of about 40 000 cycles/h in a coupled substrate assay. 4. The presence of the covalently bound coenzyme made the active sites difficult to inhibit with a competitive inhibitor. For example, 10 mM AMP inhibited the activity of the preparation by 50% whereas a reference system containing native alcohol dehydrogenase was inhibited by 80% in spite of the fact that the reference system contained about 20 000 times as high a concentration of coenzyme.
The analogues of the coenzyme NADP+, nicotinamide--8-bromo-adenine dinucleotide phosphate (Nbr8ADP+) and 3-iodopyridine--adenine dinucleotide phosphate (io3PdADP+), were prepared. Nbr8ADP+ was found to be active in the hydrogen transfer adn io3PdADP+ is a coenzyme competitive inhibitor for 6-phosphogluconate dehydrogenase. The binding of NADP+, NADPH and NADPH together with 6-phosphogluconate as well as that of both analogues to crystals of the enzyme 6-phosphogluconate dehydrogenase has been investigated at 0.6-nm resolution using difference electron density maps. The molecules bind in a similar position in a cleft in the enzyme subunit distant from the dimer interface. The orientation of the coenzyme in the site has been determined from the io3PdADP+ -NADP+ difference density. The ternary complex difference density extends beyond that of the nicotinamide moiety of the coenzyme and tentatively indicates substrate binding. No clear identification of the bromine atom of Nbr8ADP+ can be made. However, the analogue is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and has thus been used to fit a molecular model using an interactive graphics system, checking for preferred geometry. A possible conformation is presented which is significantly different from that of NAD+ in the lactate dehydrogenase ternary complex.
Normal duck erythrocytes and erythrocytes infected with Plasmodium lopharae have all of the enzymes for coenzyme A biosynthesis, whereas parasites freed from their host cells have non. Since erythrocytefree cultivation of P. lophurae requires an exogenous source of coenzyme A, this parasite must obtain its coenzyme A entirely from the host cell during infection.
The techniques of fluorescence enhancement, fluorescence quenching, fluorescence polarization, and equilibrium dialysis are utilized to study the binding properties of coenzyme to horse liver alcohol dehydrogenase. Polarization of fluorescence and equilibrium dialysis show that NADH binds to alcohol dehydrogenase with a stoichiometry of 6 mol per mol of enzyme, in contrast to the value of 2 determined from fluorescence enhancement measurements. NAD+ also binds with a stoichiometry of six as was determined by equilibrium dialysis. The two NADH sites which bind coenzyme more tightly and which are revealed by fluorescence enhancement measurements are designated the catalytic sites. Binding of coenzyme to the four ancillary sites does not alter the quantum yield of NADH but results in a 20% contribution to quenching of enzyme's tryptophan fluorescence. From the emission anisotropy of bound NADH of 24.0% for the additional sites and 28.1% for the catalytic sites and their relative fluorescence lifetimes at the same wavelengths of excitation and emmision, we conclude that the nicotinamide ring of NADH bound to the additional sites exhibits a freedom of motion independent of the macromolecule, while that bound to the catalytic sites is more rigidly held. Polarization of fluorescence yields negative intrinsic free energies of 9.2 and 7.5 Cal M-1 for NADH interaction with the catalytic and additional sites, respectively. Although these values are 1.3 to 2.0 Cal higher than those determined by fluorescence quenching and equilibrium dialysis, the mean Hill coefficient of 1.76 plus or minus 0.06, the titration span of 2.4 logarithmic units and coupling free energies (in magnitude and sign) are the same for all these techniques. The above difference in the intrinsic free energies are attributed largely to the different modes of interaction of excited and unexcited NADH molecules with alcohol dehydrogenase.