Activity Of Coenzyme Research Articles

Evidence for existence and a tentative identification of coenzyme in yeast thiamine pyrophosphokinase

Thiamine pyrophosphokinase (E.C. 2.7.6.2.) from Saccharomyces cerevisiae was found to require the presence of a non-protein, non-metal compound for its activity. myo -Inositol was found capable of stimulating the kinase activity in the presumably resolved but otherwise crude sample of the enzyme. The hexytol was also found capable of inducing the enzyme in growing yeast cells. The cultured yeast cells, in which the kinase had been induced, were used as source of the enzyme for its purification. The compound that had been left adsorbed to the final column of DEAE-Sephadex was proved to have a coenzyme activity towards the enzyme and tentatively identified with myo -inositol 1-pyrophosphate. A sample of synthetic myo -inositol 1-pyrophosphate was made and its coenzyme activity was observed.

Preparation of analogues of ATP, ADP and AMP suitable for binding to matrices and the enzymic interconversion of ATP and ADP in solid phase.

Alkylation of ATP with iodoacetic acid at pH 6.5 yielded 1-carboxymethyl-ATP which, after alkaline rearrangement, gave N-6-carboxymethyl-ATP. Condensation of this analogue with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated N-6-[(6-aminohexyl)carbamoylmethyl]-ATP in an overall yield of 40% based on the parent nucleotide ATP. The coenzymic activities of both N-6-adenine-substituted derivatives of ATP were tested with three kinases. Both derivatives showed coenzymic function against hexokinase with the "long" derivative having highest activity (95%) relative to unsubstituted ATP. Their activities towards the other two kinases tested was negligible except with the "long" analogue against glycerokinase (20%). The latter ATP analogue, when bound to Sepharose through its terminal amino group, could be dephosphorylated to the corresponding ADP analogue with soluble hexokinase yielding glucose 6-phosphate in an enzymic "solidphase" fashion. The Sepharose-bound ADP formed could subsequently be phosphorylated back to ATP using soluble acetate kinase. Sepharose-ATP preparations were also used in preliminary affinity chromatography studies using citrate synthase. Alkylation of ADP following the above procedure yielded the corresponding ADP analogue, N-6-[(6-aminohexyl)carbamoylmethyl]-ADP in an overall yield of 40%. Alkylation of AMP yielded the corresponding N-6-[(6-aminohexyl)carbamoylmethyl]-AMP in an overall yield of 45%.

Ethanolamine ammonia-lyase: Inactivation of the holoenzyme by N 2O and the mechanism of action of coenzyme B 12

Functional ethanolamine ammonia-lyase is inactivated by N 2O as well as by O 2, indicating that the active form of coenzyme B 12 is an enzyme-bound corrin derivative in which the CoC bond of the coenzyme is broken and the cobalt ion is in the +1 state of oxidation. The nucleoside fragment formed in the process of coenzyme activation is tentatively identified as 4′,5′-didehydro-5′-deoxyadenosine. A mechanism of action of ethanolamine ammonia-lyase is formulated in analogy to that of DL-1,2-propanediol dehydrase and compared to proposed alternative reaction schemes.

Demonstration of cobamide coenzyme in Ascaris suum

1. 1. Cobamide coenzyme was extracted from the body wall of adult female Ascaris suum maintained in Ellison's medium containing vitamin B 12. 2. 2. The identification of the coenzyme in purified fractions was based on (a) specific spectral changes following treatment of the preparation with light and cyanide, (b) coenzyme activity in the glutamate mutase reaction and (c) inactivation of the coenzyme activity by light. 3. 3. A fivefold increase in coenzyme occurred in worms maintained in Ellison's medium plus vitamin B 12, compared to the endogenous value. The data indicate that vitamin B 12 is converted by A. suum into the metabolically active coenzyme.

Studies on new intracellular proteases in various organs of rat. 2. Mode of limited proteolysis.

1. The mechanism of proteolysis of ornithine transaminase apoenzyme II by group-specific protease and the relation between the confirmations of ornithine transaminase and its susceptibility to group-specific protease were studied to elucidate the mode of action of the protease. 2. Differences in the conformations of ornithine transaminase apoenzyme II, molecular weight 67000, and ornithine transaminase holoenzyme, molecular weight 140000, were shown by studies on difference spectra produced by various concentrations of ethylene glycol. Increase of the titratable sulfhydryl groups on resolution of the coenzyme from ornithine transaminase also supports this finding. These results are consistent with the facts that the apoenzyme was sensitive to group-specific protease, while the holoenzyme was not. 3. Kinetics studies showed that ornithine transaminase apoenzyme II was degraded by limited proteolysis. Reaction of the native enzyme with group-specific protease resulted in a nick in the enzyme molecule with formation of one homogeneous large product and small peptides. The large product was not degraded further. The large product was indistinguishable from native ornithine transaminase apoenzyme II in various properties including its elution volume on gel filtration, its mobility on disc electrophoresis, its antigenicity, its estimated number of exposed tryptophan residues, and its titratable number of sulfhydryl groups. But unlike the apoenzyme the product did not show tetramerization with coenzyme or catalytic activity, although it retained the ability to bind with coenzyme and had the same number of bound pyridoxal phosphate as the native ornithine transaminase molecule. Thus, native ornithine transaminase apoenzyme II was degraded by limited proteolysis. Unfolded enzyme, denatured by 8 M urea, was degraded extensively. 4. The initial step of intracellular proteins degradation is discussed on the basis of these results.

Nicotinamide 3,N4-ethenocytosine dinucleotide, an analog of nicotinamide adenine dinucleotide. Synthesis and enzyme studies

A structural analog of NAD+, NICOTINAMIDE 3,N-4ethenocytosine dinucleotide (epsilonNCD+), has been synthesized, characterized, and compared in activity with the natural coenzyme in several enzyme systems. The Vmax and apparent Km values were determined for NAD+, epsilonNCD+, and epsilonNAD+ (nicotinamide 1, N6-ethenoadenine dinucleotide) with yeast alcohol, horse liver alcohol, pig heart malate, beef liver glutamate, and rabbit muscle lactate and glyceraldehyde-3-phosphate dehydrogenases. The Vmax for epsilonNCD+ was as great or greater than that obtained for NAD+ with three of the enzymes, 60-80 per cent with two others, and 14 percent with one. EpsilonNCD+ was found to be more active than epsilonNAD+ with all six dehydrogenases. EpsilonNCD+ served as a substrate for Neurospora crassa tnadase, but could not be phosphorylated with pigeon liver NAD+ kinase. NAD+ pyrophosphorylase from pig liver was unable to catalyze the formation of epsilonNCD+ from the triphosphate derivative of epsilon-cytidine and nicotinamide mononucleotide, but was able to slowly catalyze the pyrolytic cleavage of epsilonNCD+. The coenzyme activity of epsilonNCD+ with dehydrogenases can be discussed in terms of the close spatial homology of epsilonNCD+ and NAD+, which may allow similar accommodations within the enzyme binding regions.

Lipid synthesis in stringent Escherichia coli: an artifact in acetate labeling of phospholipids during a shiftdown in growth rate.

The use of (14C)acetate to label the phospholipids of stringent Escherichia coli, after a decrease in agitation, leads to artifacts resulting from a decrease in the specific activity of the acetyl coenzyme A pool.

The synthesis of adenine-substituted derivatives of NADP and their potential as active coenzymes and affinity adsorbents.

Alkylation of NADP+ with iodoacetic acid at pH 6.5 yielded N1‐carboxymethyl‐NADP+ which, following enzymic reduction, alkaline rearrangement and enzymic reoxidation, gave N6‐carboxy‐methyl‐NADP+. Condensation of these analogues with 1,6‐diaminohexane in the presence of a water‐soluble carbodiimide generated the N1 and N6N‐(6‐aminohexyl)‐acetamide derivatives of NADP+ respectively. The coenzymic activities of the N1 and N6 adenine‐substituted carboxymethyl and N‐(6‐aminohexyl)‐acetamido derivatives of NADP+ were tested with several NADP+‐dependent dehydrogenases. The carboxymethyl derivatives were more active (up to 145%) relative to un‐substituted NADP+ than the corresponding N‐(6‐aminohexyl)‐acetamido compounds (up to 10%).NADP+ ‐N6‐[N‐(6‐aminohexyl)‐acetamide] was coupled to a soluble dextran and to Sepharose 4B by the cyanogen bromide technique. The dextran‐bound derivative contained 85 μmol NADP+ analogue/g dry dextran and was reduced at approximately 35% the rate of native NADP+ with glucose‐6‐phosphate dehydrogenase. The dextran‐bound NADP+ cycled at a rate of about 30% relative to free NADP+ in a system comprising glucose‐6‐phosphate dehydrogenase and l‐glutamate dehydrogenase. Sepharose‐bound NADP+ proved to be an effective biospecific adsorbent for affinity chromatography and was competent for the resolution of a mixture of bovine albumin, lactate dehydrogenase and glucose‐6‐phosphate dehydrogenase. The latter was eluted almost quantitatively with a pulse of 0.5 mM NADP+.

The microdetermination of 2,3-diphosphoglycerate

A procedure for microestimation of 2,3-diphosphoglycerate, utilizing its role as coenzyme in the phosphoglycerate mutase reaction is described. The coenzymic activity was determined by assaying phosphoglycerate mutase polarimetrically without a coupled enzyme. This method is applicable to samples containing as little as 0.002 μmole of 2,3-diphosphoglycerate/ml. The content in various biological extracts was determined.

Characterization of branched chain fatty acids from subcutaneous triacylglycerols of barley-fed lambs.

AbstractThe fatty acids of subcutaneous triacylglycerols (containing ca. 11% of branched chain components) from lambs fed on barley‐rich diets were fractionated by treatment with mercuric acetate and by urea adduct formation to yield concentrates rich in the branched chain components, all of which were saturated. The concentrates were subjected to analysis by high resolution gas liquid chromatography in conjunction with mass spectrometry. The branched chain fatty acids consisted of a complex mixture of mono‐, di‐, and trimethyl substituted components. The greater part of the mixture comprised monomethyl substituted acids of chain length 10–17 carbon atoms. Within each of these molecular species, a number of positional isomers was identified, notably in respect of methyltetradecanoic acid (methyl substituent on carbon 2, 4, 6, 8, 10, and 12) and methylhexadecanoic acid (methyl substituent on carbon 2, 4, 6, 8, 12, and 14). Homologous series also could be recognized of one of which all eight members from 4‐methyldecanoic acid to 4‐methylheptadecanoic acid were identified; together they accounted for ca. 39% of the branched chain fatty acids which were sampled for mass spectrometry. The dibranched acids identified consisted of five members of a homologous series, ranging in chain length from 11–15 carbon atoms and with substituent methyl groups at positions 4 and 8. Though the identity of only one tribranched acid (2,6,10‐trimethyltetradecanoic acid) was established, others also apparently were present in the mixture. The probable involvement of methylmalonate in the biosynthesis of these branched chain acids is discussed briefly, with particular reference to the availability of vitamin B12 in relation to the activity of methylmalonyl coenzyme A mutase.

Relationships between coenzyme Q and vitamin E.

Coenzyme Q and vitamin E have limited organic structural features in common, but their biochemical ce:roles in life processes can be distinctly different. Vitamin E is a chromanol; the quinone analog of vitamin E related to coenzyme Q is a plastoquinone which has no ce:role in nutrition, but functions in photosynthesis. Coenzyme Q is a quinone and its chromanol, similar to vitamin E, has no ce:role in life processes. The organic chemical and biochemical differentiation between coenzyme Q and vitamin E were less clear in the early 1960’s, but fortunately led to a study of coenzyme Q in experimental animal diseases caused by diets low in vitamin E. These studies demonstrated the lifesaving and vitamin activity of coenzyme Q in rabbits, monkeys, rats, hamsters, chickens, and turkeys. Coenzyme Q has indispensable, electron transfer functions in three mitochondrial oxidases, and functions in the Golgi apparatus, and is a component of a cytoplasmic aldehyde oxidase. Vitamin E does not coenzymatically substitute for coenzyme Q in any CoQ10-enzyme which is in agreement with their organic structural differences. The biosynthesis of coenzyme Q10, in human tissue requires many of the known vitamins and essential minerals and underscores the importance of coenzyme Q in nutritional sciences.

A new immobilized NAD+ analogue, its application in affinity chromatography and as a functioning coenzyme.

Alkylation of NAD+ with iodoacetic acid followed by alkaline rearrangement gave N6‐carboxymethyl‐NAD+. Condensation of this analogue with 1,6‐diaminohexane gave the analogue NAD+‐ N6‐[N‐(6‐aminohexyl)‐acetamide]. The coenzymic activity of the two derivatives was tested with malate dehydrogenase, alcohol dehydrogenase and lactate dehydrogenase. The efficiency relative to unsubstituted NAD+ was in the range of 50–100%.NAD+‐N6‐[N‐(6‐aminohexyl)‐acetamide] was attached to Sepharose 4B by the cyanogen bromide method. The immobilized NAD+ analogue thus obtained, exhibited cofactor activity when tested in a recycling three‐enzyme system (malate dehydrogenease‐citrate synthase‐lactate dehydrogenase). The immobilized NAD+ analogue proved to be and effective ligand in affinity chromatography. Thus a mixture of albumin, alcohol dehydrogenase and lactate dehydrogenase was resolved with good recovery. The enzymes were eluted with NAD+ and NADH, respectively.

Mechanism of coenzyme activity of pyridoxal 5′ - phosphate hydrazones

Summary Pyridoxal 5′-phosphate hydrazones and pyridoxal 5′-phosphate (PLP) behave similarly towards PLP-dependent enzymes. The work described here shows that these hydrazones are dissociated under the conditions of the enzymatic reaction to yield free PLP which accounts for their apparent coenzyme activity. It is shown that PLP-dependent enzymes do not catalyze the dissociation of hydrazones, but that the aminogroups of the substrates do promote this dissociation. These results indicate that no contradiction exists between the apparent coenzyme activity of PLP hydrazones and the widely accepted mechanism of PLP catalysis involving a free aldehyde group.

The Effect of Vitamin B12 Deprivation on the Enzymes of Fatty Acid Synthesis

The enzymes of fatty acid synthesis from liver and brain in normal and B12-deprived rats were studied. Both total and specific activities of fatty acid synthetase and acetyl coenzyme A carboxylase were 2- to 5-fold greater in B12 deprivation than in the normal state. The presence of excess activators in the B12-deprived rat or an excess inhibitor in the normal rat was not found by serial admixtures of the respective cytosol preparations or by partial purification of the enzymes. Since B12 deficiency is associated with an increase in the tissue concentrations of propionic and methylmalonic acid, the effect of the coenzyme A derivatives of these acids on fatty acid synthetase and acetyl-CoA carboxylase activity was studied. Propionyl-CoA was a substrate for fatty acid synthetase, while methylmalonyl-CoA markedly inhibited synthetase activity. Methylmalonyl-CoA markedly inhibited acetyl-CoA carboxylase activity. Propionyl-CoA was shown to be a substrate for acetyl-CoA carboxylase, competing with acetyl-CoA, and the product synthesized was methylmalonyl-CoA. Thus, in vitamin B12 deprivation propionyl-CoA competes with acetyl-CoA as substrate providing a mechanism for odd chain fatty acid production, and its product, methylmalonyl-CoA, may function as an inhibitor of the enzymes of fatty acid synthesis.

Comparison of coenzymatic activities of 2-nor-2-hydroxymethyl pyridoxal 5′-phosphate for mitochondrial and cytoplasmic aspartate aminotransferases

2-Nor-2-hydroxymethyl pyridoxal 5′-phosphate served as a somewhat superior coenzyme to pyridoxal 5′-phosphate for cytoplasmic and mitochondrial aspartate aminotransferases in the usual assay system containing Tris-HCl buffer. In the presence of phosphate anion, however, the affinities of the analog for both enzymes and the V values of the reactions mediated by the cytoplasmic and mitochondrial aspartate aminotransferases reconstituted with the analog were more markedly decreased than in the cases of the native coenzyme. Various Vitamin B 6 enzymes tested hitherto were classified into at least three groups according to their attitudes toward the coenzyme analog.

Inhibition of Rat Liver Acetyl Coenzyme A Carboxylase by N6,O2′-Dibutyryl Cyclic Adenosine 3′:5′-Monophosphate in Vitro

Abstract It has been established that hepatic lipogenesis in rats, mice, and chickens is inhibited by cyclic 3':5'-nucleotide monophosphates but their site of action has not previously been determined. Evidence is presented here that cyclic adenosine 3':5'-monophosphate (cAMP) and N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cAMP) inhibit the activity of acetyl coenzyme A carboxylase. Dibutyryl cAMP incubated with rat liver slices inhibited both lipogenesis and acetyl-CoA carboxylase activity to about the same extent. The amount of dibutyryl cAMP needed to inhibit acetyl-CoA carboxylase was in the same range as the amount needed to inhibit lipogenesis in rat and chicken liver slices. Inhibition of acetyl-CoA carboxylase by the addition of 1 mm dibutyryl cAMP to the enzyme assay reaction mixture was demonstrable even without preincubation with liver slices. It is concluded that a major portion of the inhibition of lipogenesis in rat liver by cAMP can be attributed to the inhibition of acetyl-CoA carboxylase.

On the Relationship between Fatty Acid Synthesis and the Total Activities of Acetyl Coenzyme A Carboxylase and Fatty Acid Synthetase in the Liver of Prenatal and Early Postnatal Chicks

Abstract Fructose and lactate markedly stimulated fatty acid synthesis from [1-14C]acetate in isolated cells prepared from the livers of unfed neonatal chicks (21 or 22 days of incubation). In liver cells obtained from unhatched chicks (19 days of incubation) fructose and lactate had very small stimulatory effects on fatty acid synthesis. The increase in fructose- and lactate-stimulated fatty acid synthesis during hatching was about 20-fold. Pulmonary respiration began on the 20th day and the chicks were hatched by the 21st day. Fatty acid synthesis from [1-14C]acetate (plus citrate) or [1,5-14C]-citrate in a cytosol fraction of liver increased about 20-fold during the hatching period. Concomitantly, there were 6.5- and 4-fold increases in the total activities of acetyl-CoA carboxylase and fatty acid synthetase, respectively. The effect of hatching on the total activity of fatty acid synthetase could be mimicked by incubating 18-day-old embryos in an atmosphere of 100% oxygen for 24 hours. The oxygen treatment did not affect the total activity of acetyl-CoA carboxylase or the rate of fatty acid synthesis in isolated hepatocytes (plus or minus fructose). Injecting glucose or fructose into 18-day-old embryos had no effect on the total activity of acetyl-CoA carboxylase or fatty acid synthetase. Feeding the neonatal chicks increased the total activities of both acetyl-CoA carboxylase and fatty acid synthetase. If the neonatal chicks were not fed, the total activity of acetyl-CoA carboxylase did not change for at least 3 days. In contrast, the total activity of fatty acid synthetase continued to increase during those 3 days of fasting. The total activities of both enzymes were increased when unfed neonatal chicks were given a single glucose or fructose injection. The effect was not observed until 6 hours after administration of the sugar despite the fact that the rate of fatty acid synthesis was stimulated by more than 5-fold at 1½ hours after the injection. The results suggest that acetyl-CoA carboxylase and fatty acid synthetase are not part of an operon or its eukaryote equivalent, and that changes in their total activities did not initiate changes in the rate of fatty acid synthesis.

Regulation of Fatty Acid Synthesis in the Liver of Prenatal and Early Postnatal Chicks: HEPATIC CONCENTRATIONS OF INDIVIDUAL FREE FATTY ACIDS AND OTHER METABOLITES

Abstract A normal mash diet or a single glucose injection stimulates fatty acid synthesis and increases the total activities of acetyl coenzyme A carboxylase, fatty acid synthetase, and malic enzyme in the livers of neonatal chicks. Feeding and glucose injection caused a decrease in the concentration of free fatty acids and fatty acyl-CoA and an increase in α-glycerophosphate and free CoA. These concentration changes are appropriate for intermediates which may regulate both the rate of fatty acid synthesis and the concentration of the lipogenic enzymes. The hepatic concentrations of citrate and acetyl-CoA were unaffected by feeding or glucose injection. During the hatching process, total activities of acetyl-CoA carboxylase and fatty acid synthetase in the liver increased but the rate of fatty acid synthesis did not. Concomitantly, free stearate in the liver decreased, but none of the other metabolites indicated above were affected. Fatty acid synthetase increased and hepatic free stearate decreased when embryos were incubated in 100% oxygen. Stearate or a metabolite derived from stearate is a potential negative regulator of the concentration of fatty acid synthetase in chick liver. Injection of glucose into 18-day-old embryos markedly increased the α-glycerophosphate content of the liver but had no effect on the total activities of the lipogenic enzymes. Hence, α-glycerophosphate is not a direct regulator of the total activities of acetyl-CoA carboxylase or fatty acid synthetase in embryonic chicks.

Mechanism by which hydnocarpic acid inhibits mycobacterial multiplication.

Recent work in this laboratory has shown that hydnocarpic acid (HA), a principal constituent of chaulmoogra oil, inhibits multiplication in vitro of a number of mycobacterial species. This activity of HA was not shared by several straight-chain fatty acids and by dihydrochaulmoogric acid. A study of the interaction of HA with biotin has been undertaken, based on a structural analogy between biotin and the cyclopentenyl fatty acid. The multiplication of a strain of Mycobacterium intracellulare susceptible to 2 mug of HA/ml was measured turbidimetrically in Dubos medium, in the presence and absence of biotin and several other compounds. Biotin and, to a lesser extent, adenine plus guanine, palmitic acid, and linoleic acid antagonized growth inhibition by HA. Desthiobiotin, thioctic acid, and succinic acid did not block inhibition of bacterial multiplication by HA. HA may act by blocking the coenzymatic activity of biotin, or it may inhibit microbial biotin synthesis. Resumption of multiplication of M. intracellulare after a period of inhibition by HA in broth culture was found to be accompanied by reduction of the effective concentration of the drug; this could have resulted from metabolism of HA or production of an antagonist to HA by the organisms. Also, those organisms that multiplied in the presence of HA were found to represent HA-resistant mutants of M. intracellulare.

Coenzymatic activities of 2-nor-2-hydroxymethyl pyridoxal 5'-phosphate for various vitamin B6 enzymes.

The coenzymatic activities of 2-nor-2-hydroxymethyl-pyridoxal 5'-phosphate (2'-hydroxy PLP) for various vitamin B6-dependent enzymes were studied in order to obtain information on the role of the methyl group at position 2 of pyridoxal 5'-phosphate (PLP) for the binding to apoprotein and for the catalytic action of the holoenzyme. Although it has been demonstrated that this coenzyme analog binds to the catalytic center and serves as a coenzyme for a variety of B6 en-zymes, the mode of binding as well as the catalytic activity of the holo-enzyme reconstituted with the analog was different according to the kinds of enzyme and assay conditions. In the ordinary assay system using Tris-HCl buffer, 2'-hydroxy PLP showed almost an equal affinity for pig heart muscle mitochondrial and cytoplasmic aspartate aminotransferase (GOTs, and GOTm) to that of PLP. The maximal velocity (Vmax value) of the reaction mediated by GOTs, or GOTm reconstituted with 2'-hydroxy PLP was somewhat large as compared with that catalyzed by the native holoenzyme. How-ever, when phosphate buffer was used for the assay system, the affinity of the coenzyme analog decreased markedly and the Vmax value catalyzed by the analog-bound GOTs, or GOTm became approximately 70% of that by the native enzyme. Thus as judged from the Vmax value under ordinary assay conditions, 2'-hydroxy PLP is considered to be rather superior to PLP as the coenzyme for GOT. Phosphate anion inhibited the binding of PLP or 2'-hydroxy PLP to apoGOT in a competitive manner and further caused an appreciable decrease in the apparent Vmax of the reaction catalyzed by the resulting holoenzyme. The K1, values of phosphate anion against PLP and 2'-hydroxy PLP were 11.9 and 3.75, respectively. On the other hand, in the case of E. coli tryptophanase, both the affinity for 2'-hydroxy PLP and the Vmax value of the reaction mediated by the analog-bound enzyme were significantly smaller than those of the native coenzyme irrespective of the buffer used. Of the other B6 enzymes tested hitherto, E. intermedia tyrosine phenol-lyase and Pseudomonas dacunhae aspartate β-decarboxylase showed the same behaviors toward 2'-hydroxy PLP as that of tryptophanase. On the contrary, the coenzyme analog served as a more efficient co-enzyme than the native coenzyme for E. coli D-serine dehydrase in the presence of phosphate anion. These results indicate that there are at least three groups of B6 enzymes exhibiting different interaction with the 2-methyl group of PLP.