Enzymatic Decarboxylation Research Articles

Biosynthesis of porphyrins and corrins.

Haem, chlorophyll and vitamin B12 are all derived ultimately from four molecules of the pyrrole porphobilinogen (PBG) and the initial enzyme catalysed condensation of PBG leads to the unsymmetrical type III isomer of uroporphyrinogen. On the basis of straightforward chemical considerations the type I isomer should be formed and so the porphyrinogen-forming enzymes of all living systems must catalyse a highly specific rearrangement process. The nature and chemical mechanism of this rearrangement poses one of the most fascinating problems in the porphyrin field and so it is not surprising that over 20 hypothetical schemes have been proposed to account for it. Analysis of the problem suggested that the incorporation of doubly 13C-labelled precursors into the rearranged macrocyclic rings would give valuable new information on the nature of the rearrangement process. In this approach the meso=bridge atoms are of crucial importance, and several unambiguous syntheses of 13C-labelled pyrroles and porphyrins were developed to allow rigorous n.m.r. assignments to be made, and also to provide substrates for enzymic experiments. Studies carried out with enzymes from both avian blood and from Euglena gracilis have revealed the precise nature of the assembly of four PBG molecules into the type-III macrocycle: it is the same in both systems despite their vastly different evolutionary development. Complementary studies are in progress in order to determine the intermediates involved in the conversion of PBG into uroporphyrinogen III. The synthesis of amino methyl pyrromethanes and their interaction in the presence of PBG with the appropriate enzyme systems are described. It is important for the work to be able to separate not only isomeric pyrromethanes but also the four isomeric coproporphyrins. Powerful methods are described which make use of high pressure liquid chromatography for both types of separation process. Once uroporhyrinogen III has been built enzymically, there is a stepwise enzymic decarboxylation of the four acetic acid residues. A heptacarboxylic porphyrin shown to be a type-III porphyrin is isolated from the action of avian blood enzymes on porphobilinogen. Spectroscopic studies with 13C-labelling limit the possible structures to two and total synthesis of these substances shows that the natural product carries its methyl group on ring D. An isomeric heptacarboxylic porphyrin having its methyl group on ring C is of particular interest in relation to the biosynthesis of vitamin B12. This substance is synthesized together with uroporphyrin III, 14C-labelled specifically in ring C. This latter product is used to settle one of the key questions concerning nature's route to vitamin B12 - that is, does the corrin macrocycle arise from uroporphyrinogen III? Incorporation studies and specific degradations prove specific incorporation of uroporphyrinogen III into cobyrinic acid, which is the known precursor of vitamin B12.

Pyridoxal-5-phosphate levels in rat brain assayed by a modified method using enzymic decarboxylation of L-[14C]tyrosine.

Journal of NeurochemistryVolume 26, Issue 2 p. 405-407 Pyridoxal-5-phosphate levels in rat brain assayed by a modified method using enzymic decarboxylation of l-[14C] tyrosine R. A. Bayoumi, R. A. Bayoumi Courtauld Institute of Biochemistry Middlesex Hospital Medical School, London W1P 5PR, U.K.Search for more papers by this authorW.R.D. Smith, W.R.D. Smith Courtauld Institute of Biochemistry Middlesex Hospital Medical School, London W1P 5PR, U.K.Search for more papers by this author R. A. Bayoumi, R. A. Bayoumi Courtauld Institute of Biochemistry Middlesex Hospital Medical School, London W1P 5PR, U.K.Search for more papers by this authorW.R.D. Smith, W.R.D. Smith Courtauld Institute of Biochemistry Middlesex Hospital Medical School, London W1P 5PR, U.K.Search for more papers by this author First published: February 1976 https://doi.org/10.1111/j.1471-4159.1976.tb04494.xCitations: 8Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume26, Issue2February 1976Pages 405-407 RelatedInformation

Potentiometric determination of L-glutamic and pyruvic acids via enzymatic decarboxylation

A potentiometric method, for the determination of L-glutamic acid via enzymatic decarboxylation, was reported previously. In this method, a CO 2-electrode is used for the measurement of carbon dioxide released from the decarboxylation of glutamic acid. The experiments herein described indicate significant improvement in both linearity and response time. The enzyme was dispersed in either phosphate buffer or in an acidified KREBS-RINGER bicarbonate buffer (pH 3.65, i.e. the optimum acidity of the decarboxylase) at 37°C. A nernstian relationship was observed between the CO 2-electrode potential and the glutamic acid concentration in the physiological range. The response times was reduced to one and one-half minutes from the ten to twenty minutes previously reported. In addition to the improved glutamic acid study, we have applied this method to the determination of pyruvic acid, an α-keto acid. Again, a nerstain relationship was observed.

Biosynthesis of porphyrins and related macrocycles. Part VIII. Enzymic decarboxylation of uroporphyrinogen-III: structure of an intermediate, phyriaporphyrinogen-III, and synthesis of the corresponding porphyrin and of two isomeric porphyrins

A porphyrin formed when porphobilinogen (1) is incubated with an enzyme system from avian erythrocytes is proved to be heptacarboxylic and of the type III series; its properties correspond with those of phyriaporphyrin-III. Two of the four possible structures for this porphyrin are eliminated by preparing it from [2,11-13C2]porphobilinogen and carrying out a 13C n.m.r. analysis of the product, with use of a praseodymium shift reagent.Synthesis of all four heptacarboxylic porphyrins shows that the natural product has undergone specific decarboxylation of the acetic acid side chain on ring D to form (after aromatisation) the ring D methylporphyrin [as (12)]. Our results are consistent with the view that the initial enzymic decarboxylation on the pathway from uroporphyrinogen-III (2) to coproporphyrinogen-III (4) occurs at ring D.Studies are outlined leading to effective large-scale syntheses of key pyrrolic building blocks for this and other work and several methods of general interest in this area are reported.

ラット脳および腎L-芳香族アミノ酸脱炭酸酵素による3，4-DihydroxyphenylserineからのNorepinephrineの生成

The enzymic decarboxylation of stereoisomers of DOPS was examined using rat brain and kidney decarboxylase. Optimal assay conditions for racemic threo- and racemic erythro-DOPS decarboxylation were determined by the experiments concerning 1) time course, linear for 20 min, 2) optimal pH, pH 8.2, 3) optimal temperature, 37 degrees C except racemic L-threo-DOPS decarboxylation by kidney enzyme and 4) protein concentration, 1 to 5 mg in incubation medium. Under the optimal assay condition, Km of brain enzyme for L-threo-DOPS was 1.43 X 10(-3)M and Vmax, 2.22 nmoles NE/mg/15min, and Km for racemic erythro-DOPS was 10(-3)M and Vmas, 4.3 nmoles NE/mg/15min. Km of kidney enzyme for L-threo-DOPS was 1.37 X 10(-3)M and Vmax 21 nmoles NE/mg/15min, and Km for racemic erythro-DOPS was 8.7 X 10(-4)M and Vmax, 16.7 nmoles NE/mg/15min. On the other hand, D-threo and D-erythro-DOPS were scarcely decarboxylated. Decarboxylation of L-threo-DOPS in kidney enzyme was markedly inhibited by D-threo-DOPS. Kinetic analysis revealed that the type of inhibition was non-competitive. In helically-cut strips of isolated rabbit aorta, the contractile response to NE (10(-8)g/ml) formed from L-threo-DOPS was 95% that of 1-NE (10(-8)g/ml) while the response to NE (10(-8)g/ml) formed from racemic erythro-DOPS was not detectable. These results suggest that L-threo-DOPS is a more effective precursor of natural 1-NE than racemic threo-DOPS.

Mixed function oxidation of hexobarbital and generation of NADPH by the hexose monophosphate shunt in isolated rat liver cells

Mixed function oxidation of hexobarbital and the generation of NADPH by the hexose monophosphate shunt were studied in isolated rat liver parenchymal cells from phenobarbital-pretreated and untreated animals. In cells isolated from untreated rats, a maximal rate of hexobarbital oxidation of 17 μmol·g −1 liver wet weight·(60 min) −1 was observed, while in cells isolated from phenobarbital-pretreated rats a maximal rate of 29 μmol·g −1 liver wet weight·(60 min) −1 has been obtained. On the basis of the specific radioactivity at carbon atom 1 of glucose 6-phosphate, fructose 6-phosphate and 6-phosphogluconate, determined by enzymatic decarboxylation, a ratio between NADPH formation via the hexose monophosphate shunt and NADH utilization for hexobarbital oxidation of 6:1 in untreated and 9.5:1 in pretreated cells has been obtained. With phenazine methosulfate the stimulation of NADPH generation via the hexose monophosphate shunt exceeded that observed in the presence of hexobarbital by 329 and 160%, respectively, indicating that the capacity of this pathway is sufficient to provide more reducing equivalents than are required for maximal rates of mixed function oxidation.

Enzymic decarboxylation of s-adenosyl-l-methionine in rat liver: possible interaction of putrescine with the prosthetic group.

S-Adenosyl-L-methionine decarboxylase (EC 4.1.1.50) has been purified more than 1000-fold from rat liver. The molecular weight of the decarboxylase was calculated to be 68 000. No evidence was obtained indicating that pyridoxal phosphate acts as the prosthetic group of the enzyme. On the other hand, the decarboxylase apparently contains some carbonyl group(s) participating in the catalysis as supported by the inhibition of S-adenosyl-L-methionine decarboxylation in the presence of NaBH-4, phenylhydrazine or NaCN. Putrescine, the specific activator of mammalian S-adenosyl-L-methionine decarboxylase, might interact, directly or indirectly, with the carbonyl group(s) of the enzyme as suggested by the protection of the decarboxylase activity against borohydride reduction by the diamine.

Stereochemistry of the enzymic decarboxylation of L-lysine

The decarboxylation of L-lysine to yield cadaverine, catalysed by L-lysine decarboxylase (EC 4.1.1.18, L-lysine carboxylyase), takes place with retention of configuration.

Transaminations of amino donors with α-ketoglutarate in normal and neoplastic mouse tissues measured by a simple radiometric procedure

In the present work the estimation of aminotransferase activities with α-ketoglutaric-1- 14C as acceptor has been simplified to the extent that it is not necessary to achieve separation of α-ketoglutarate or glutamate from the reaction mixture in order to determine the extent of transamination. The determination consists of a two-step procedure. The first step is the transamination of α-ketoglutarate-1- 14C with an amino donor to produce glutamic acid-1- 14C. The second step is the enzymatic decarboxylation of the labeled glutamate and the measurement of the 14CO 2 produced. This method affords a rapid and variably sensitive procedure for multiple determinations of those aminotransferase activities in which α-ketoglutarate is the acceptor. It has been applied in our present work to the determination of aminotransferase activities of several normal mouse tissues and red cells and serum and to two transplantable mouse tumors. The amino donors employed were 19 amino acids commonly found in proteins, 17 aminophosphonic acids, and several amines and diamino monocarboxylic acids. A possible application of this method to clinical problems is mentioned.

Specific inhibition of the enzymic decarboxylation of S-adenosylmethionine by methylglyoxal bis(guanylhydrazone) and related substances.

Methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidene)-dinitrilo]diguanidine} is a very potent inhibitor of putrescine-activated S-adenosylmethionine decarboxylases from many different mammalian tissues, including sublines of mouse L1210 leukaemia that are resistant to the drug as well as sublines that are sensitive. The inhibition of purified rat ventral prostate S-adenosylmethionine decarboxylase is competitive with respect to the S-adenosylmethionine substrate, and is much greater in the presence than in the absence of the activator putrescine. Inhibition by the drug depends, among other things, on the nature of the aliphatic amines that can serve as stimulators of rat prostate S-adenosylmethionine decarboxylase. Effects of some congeners of methylglyoxal bis(guanylhydrazone) on the enzyme are described.

Studies on pyrimidine derivatives and related compounds. LXXXVII. Reaction of thiamine analogues with diethyl benzoylphosphonate.

Reaction of thiamine analogues lacking the 5-hydroxyethyl group (6a-e) with diethyl benzoylphosphonate (7) was carried out. Comparison of reactivity in 4'-substituted thiamine analogues and those lacking the hydroxyethyl group suggests the presence of an interaction in the thiamine molecule between the pyrimidine ring and the hydroxyethyl group in an aprotic solvent. Reactivity at the 2 position (the active center in enzymatic decarboxylation) in 4'-substituted thiamine analogues may be affected by this interaction.

Lipid requirement of membrane-bound ATPase. Studies on human erythrocyte ghosts.

The (Na++ K+)‐stimulated ATPase of human erythrocyte ghosts is completely inactivated by treatment with pure phospholipase A2 or phospholipase C. These phospholipases also cause considerable or even complete loss of Mg2+‐dependent ATPase activity. Enzymatic hydrolysis of sphingomyelin does not significantly influence either of the ATPase activities. Conversion of the glycero‐phospholipids into phosphatidic acid by phospholipase D causes a drastic decrease in the Mg2+‐dependent ATPase activity, whereas the (Na++ K+)‐stimulated activity is increased. Enzymatic decarboxylation of the phosphatidylserine fraction of the ghosts does not influence the (Na++ K+)‐stimulated ATPase, unless the last 13% of this fraction is converted into phosphatidyethanolamine, when there is a complete loss of this particular ATPase activity. On the other hand, the Mg2+‐dependent activity tends to be increased by this treatment. The (Na++ K+)‐stimulated ATPase activity of erythrocyte ghosts treated with phospholipase C and subsequently extracted with dry ether, can be completely reconstituted by the addition of phosphatidylserine. Phosphatidylcholine, phosphatidylethanolamine and phospholipid mixtures are found to be ineffective. Phosphatidic acid produced a partial restoration of activity. It is concluded that the (Na++ K+)‐stimulated ATPase activity strongly depends upon the presence of phosphatidylserine, of which only a minor fraction in the erythrocyte membrane is directly involved. It cannot be concluded from the experiments described in this paper that the Mg2+‐dependent ATPase also requires a specific phospholipid. However, a direct involvement of either cholesterol or sphingomyelin in this activity can be precluded.

Adenosylmethionine decarboxylase in developing rat brain.

AbstractAdenosylmethionine decarboxylase from rat brain has been found to be similar to the same enzyme isolated from other rat tissues in regard to kinetic parameters, pH optimum, putrescine requirement, and subcellular location. Evidence is presented that pyridoxal phosphate is not the functional cofactor in enzymatic decarboxylation by the rat brain preparation. The capacity for spermidine synthesis in developing rat brain was determined by measurement of the activity of adenosylmethionine decarboxylase. The activity increased dramatically after 10 days of postnatal age. This increase occurred after the period of maximum nucleic acid synthesis, an observation which suggests that spermidine may have a role in the functional development of the brain.

An automated P CO 2 assay for glutamic acid decarboxylase

An automated assay for determination of glutamic acid decarboxylase activity is described. The P CO 2 of an incubation medium is used as a measure of total CO 2 formed by enzymic decarboxylation of glutamate. Details of manifold construction, reagent composition, and data concerning the reliability of the method are presented.

Pathways of glycollate metabolism in the blue-green alga Anabaena cylindrica.

1. Exogenous glycollate was assimilated by the blue-green alga Anabaena cylindrica. 2. About 50% of the C-1 carbon of 14C-1-glycollate (i.e.25% of the total carbon) was released as 14CO2 in the dark and also in the light in the presence of DCMU. Most of the 14CO2 released in the light in the absence of DCMU was refixed. 3. Assimilation was almost completely inhibited by α-hydroxy-2-pyridinemethane sulphonic acid, an inhibitor of enzymic glycollate oxidation. Cell extracts catalyzed the oxidation of glycollate to glyoxylate at rates sufficient to account for the in vivo assimilation. 4. Isonicotinylhydrazide, an inhibitor of the conversion of glycine to serine in higher plant/green algae glycollate metabolism, did not significantly affect glycollate metabolism in A. cylindrica. Short-term labelling experiments with 14C-1-glycollate in the light and dark did not show a significant metabolism of 14C via glycine and serine. However, the enzymes for the metabolism of glyoxylate via glycine, serine and hydroxypyruvate to glycerate were demonstrated in cell extracts, although the activity of the enzyme catalyzing the metabolism of serine to hydroxypyruvate was not sufficient to account for the in vivo rate of glycollate assimilation. 5. Cell extracts catalyzed the enzymic condensative decarboxylation of glyoxylate to tartronic semialdehyde and also the enzymic reduction of tartronic semialdehyde to glycerate. The activities in extracts were sufficient to account for the total in vivo photoassimilation of glycollate. The specific activity of malate synthase was insufficient to account for the total photometabolism of glycollate but exceeded the in vivo rate in the dark. 6. On the basis of the inhibitor and kinetic experiments and in terms of the enzymes detected, it appears that in the light glycollate is metabolized mainly via glyoxylate → tatronic semialdehyde → glycerate → 3-phosphoglycerate → (glycolytic pathway) → pyruvate → alanine plus tricarboxylic acid cycle and related compounds. The bulk of the CO2 released in the light is probably refixed via the Calvin cycle. In the dark, the glyoxylate, produced from exogenous glycollate, appears to be metabolized mainly by malate synthase directly to malate.

Measurement of aromatic l-amino acid decarboxylase—a technical comment

The enzyme aromatic amino acid decarboxylase and the enzymatic decarboxylation of DOPA in tissue homogenates and physiological fluids is being extensively studied at present because of the possible involvement of this enzyme in the pathogenesis of Parkinson's disease (1) and the therapeutic use of L-DOPA (2). Determination of the enzymatic decarboxylation of DOPA involves either measurement of dopamine (3–7) or CO 2 (8–10). Since the determination of CO 2 is relatively simple and rapid, this procedure is now frequently used to estimate enzyme activity, dopamine formation, and/or DOPA disappearance. Recent studies in our laboratory have shown, however, that the measurement of the decarboxylation of DOPA can easily lead to false results.

A radiometric technique for measuring L-asparagine in picomole quantities.

A radiometric technique for measuring l-asparagine in small samples of biological origin is presented. After removal of l-glutamate and l-aspartate by enzymic decarboxylation, l-asparagine in the sample is hydrolysed to l-aspartate with l-asparaginase from Escherichia coli. The l-aspartate so generated is then enzymically transaminated with 2-oxo[1-(14)C]glutarate yielding l-[1-(14)C]glutamate. After specific chemical removal of unchanged 2-oxo[1-(14)C]glutarate, the l-[1-(14)C]glutamate generated is determined by scintillation counting of the radioactivity. This technique can measure the concentration of l-asparagine present in approx. 1mul of mouse blood, and is capable of detecting as little as 25pmol of the amide. Illustrative applications of the method are presented and discussed, including measurement of the l-asparagine content of the mouse hypophysis.

The photooxidation of glyoxylate by envelope-free spinach chloroplasts and its relation to photorespiration

Large quantities of CO 2 are released within many photosynthesizing tissues in the light by the process of photorespiration. This CO 2 arises largely from the carboxylcarbon atom of glycolate, which is synthesized in chloroplasts during photosynthesis. Glyoxylate is then produced by the glycolate oxidase reaction. The glyoxylate may be directly decarboxylated to CO 2, but some investigators believe the glyoxylate must first be converted to glycine before CO 2 is released during photorespiration. Spinach chloroplasts with their envelope membranes removed in dilute buffer solution have now been shown to carry out the oxidative decarboxylation of [1- 14C]glyoxylate, in the presence of light and manganous ions in an atmosphere containing oxygen, to yield 1 mole each of 14CO 2 and formate. Rates of enzymatic decarboxylation exceeding 50 μmoles of 14CO 2 mg chlorophyll −1 hr −1 were obtained at pH 7.6; hydrogen peroxide is probably the oxidant in the reaction. Heated chloroplasts are inactive under the standard conditions and there is an almost absolute requirement for each of the components listed above. Conditions for some other nonenzymatic decarboxylations of glyoxylate have also been described. [1- 14C]Glycine is decarboxylated by the enzymatic system at only 1% of the rate of [1- 14C]glyoxylate. Maize chloroplast preparations are much less active than spinach chloroplasts. The high rates of CO 2 produced by the spinach system directly from glyoxylate, as well as the need for light and oxygen, suggest that this reaction functions in photorespiration, and that CO 2 arises during photorespiration without glycine as a mandatory intermediate.

Occurrence and distribution of aromatic L-amino acid (L-DOPA) decarboxylase in the human brain.

Abstract—The enzymatic decarboxylation of l‐DOPA was measured in isotonic dextrose homogenates of different regions of the human brain by estimating 14CO2 evolved from tracer amounts of d l‐DOPA[carboxy1‐14C]. Enzyme activity was linear with respect to tissue concentration and time of incubation. The reaction exhibited a pH maximum at 7·0, was completely dependent upon the presence of high concentrations of pyridoxal phosphate, proceeded at the same rate in an atmosphere of air and nitrogen, and produced dopamine in addition to CO2 as a reaction product. The enzyme preparation behaved like an aromatic l‐amino acid decarboxylase: it also decarboxylated o‐tyrosine and when incubated with 5‐hydroxytryptophan, serotonin was isolated as the reaction product; but it was devoid of activity towards d‐DOPA[carboxy1‐14C]. Within the human brain, l‐DOPA decarboxylase was most active in the putamen and caudate nucleus; the pineal gland, hypothalamus, and the reticular formation and dorsal raphe areas of the mesencephalon exhibited considerable activity. Areas of cerebral cortex exhibited very low enzymatic activity and in regions composed predominantly of white matter, l‐DOPA decarboxylase activity was not significantly above blank values. The activity of l‐DOPA decarboxylase in the human putamen and caudate nucleus tended to decrease with the age of the patients; in comparatively young subjects (46 yr old) the enzyme activity compared favourably with that found, by means of the same assay technique, in the caudate nucleus of the cat.

α-Ketobutyrate decarboxylase activity in Rhizoctonia solani

The anaerobic decarboxylation of α-ketoacids by a crude extract of Rhizoctonia solani was investigated using a manometer procedure. The possible importance of this enzyme in the metabolism of the fungus is discussed. The best substrate for the enzyme preparation was α-ketobutyrate. Thiamine pyrophosphate and Mg2+ were cofactors for the enzymic decarboxylation of α-ketobutyrate. The Km for α-ketobutyrate was 7.6 × 10−3 moles/1, and the energy of activation was 3980 calories. Optimum catalysis for the reaction was achieved at 32°, pH 6.2. Products of the action of the enzyme preparation on α-ketoacids were CO2 and the respective aldehyde. Decarboxylase activity was inhibited in the presence of Hg2+, Ag+, and norjavanicin.