4-carbon Acid Research Articles

2-Oxo-3-alkynoic Acids, Universal Mechanism-Based Inactivators of Thiamin Diphosphate-Dependent Decarboxylases: Synthesis and Evidence for Potent Inactivation of the Pyruvate Dehydrogenase Multienzyme Complex

A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl substituent at carbon 4 was reported by the authors as potent irreversible and mechanism-based inhibitors of the thiamin diphosphate- (ThDP-) dependent enzyme pyruvate decarboxylase [Chiu, C.-F., & Jordan, F. (1994) J. Org. Chem. 59, 5763-5766]. The method has been successfully extended to the synthesis of the 4-, 5-, and 7-carbon aliphatic members of this family of compounds. These three compounds were then tested on three ThDP-dependent pyruvate decarboxylases: the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) and its E1 (ThDP-dependent) component, pyruvate oxidase (POX, phosphorylating; from Lactobacillus plantarum),and pyruvate decarboxylase (PDC) from Saccharomycescerevisiae. All three enzymes were irreversibly inhibited by the new compounds. The 4-carbon acid is the best substrate-analog inactivator known to date for PDHc, more potent than either fluoropyruvate or bromopyruvate. The following conclusions were drawn from extensive studies with PDHc: (a) The kinetics of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is time- and concentration-dependent. (b) The 4-carbon acid has a Ki 2 orders of magnitude stronger than the 5-carbon acid, clearly demonstrating the substrate specificity of PDHc. (c) The rate of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is enhanced by the addition of ThDP and MgCl2. (d) Pyruvate completely protects E1 and partially protects PDHc from inactivation by 2-oxo-3-butynoic acid. (e) E1 but not E2-E3 is the target of inactivation by 2-oxo-3-butynoic acid. (f) Inactivation of E1 by 2-oxo-3-butynoic acid is accompanied by modification of 1.3 cysteines/E1 monomer. The order of reactivity with the 4-carbon acid was PDHc > POX > PDC. While the order of reactivity with PDHc and POX was 2-oxo-3-butynoic acid > 2-oxo-3-pentynoic acid > 2-oxo-3-heptynoic acid, the order of reactivity was reversed with PDC.

Expansion of C4 ecosystems as an indicator of global ecological change in the late Miocene

THE most common and the most primitive pathway of the three different photosynthetic pathways used by plants is the C3 pathway, or Calvin cycle, which is characterized by an initial CO2 carboxylation to form phosphoglyceric acid, a 3-carbon acid. The carbon isotope composition (δ13C) of C3 plants varies from about −23 to −35%l–3 and averages about −26%. Virtually all trees, most shrubs, herbs and forbs, and cool-season grasses and sedges use the C3 pathway. In the C4 pathway (Hatch–Slack cycle), CO2 initially combines with phosphoenol pyruvate to form the 4-carbon acids malate or aspartic acid, which are translocated to bundle sheath cells where CO2 is released and used in Calvin cycle reactions1–4. The carbon isotope composition of C4 plants ranges from about −10 to −14%, averaging about −13% for modern plants1–3. Warm-season grasses and sedges are the most abundant C4 plants, although C4 photosynthesis is found in about twenty families5. The third photosynthetic pathway, CAM, combines features of both C3 and C4 pathways. CAM plants, which include many succulents, have intermediate carbon isotope compositions and are also adapted to conditions of water and CO2 stress. The modern global ecosystem has a significant component of C4 plants, primarily in tropical savannas, temperate grasslands and semi-desert scrublands. Studies of palaeovegetation from palaeosols and palaeodiet from fossil tooth enamel indicate a rapid expansion of C4 biomass in both the Old World and the New World starting 7 to 5 million years ago. We propose that the global expansion of C4 biomass may be related to lower atmospheric carbon dioxide levels because C4 photosynthesis is favoured over C3 photosynthesis when there are low concentrations of carbon dioxide in the atmosphere.

Bioautography of carnitine derivatives: Compounds in human semen possessing biological activity for a carnitine-requiring mutant of the yeast, Torulopsis bovina

Paper chromatography, descending, using the solvent system: n-butanol: acetic acid: water (8: 1: 1. v/v) separated semen components which supported growth of a carnitine-requiring mutant of the yeast. Torulopsis bovina. These chromatographic zones contained free L-carnitine. its acetyl and propionyl esters, esters with 4-carbon acids and the amino acid arginine.

Simultaneous occurrence of C3 and C4 photosyntheses in relation to leaf position in Mollugo nudicaulis

SINCE the discovery of C4 photosynthesis1,2 higher plants have been categorised into C3 plants, which form 3-phosphoglycerate (PGA) as the first labelled intermediate, and C4 plants, which form 4-carbon acids as the predominant early photosynthetic products, C4 plants can also be distinguished by features such as Kranz leaf anatomy, low photorespiration, high photosynthetic efficiency and reduced 13C discrimination3. Although 943 C4 plants are known to be distributed among 18 families, only 18 genera from 10 families include both C3 and C4 plants4. Kennedy and Laetsch5 described Mollugo verticillata as intermediate for C3 and C4 photosynthesis. But Brown and Brown6 felt that it was a typical C3 plant. Recently three Panicum species (P. milioides, P. hians and P. laxum) have been found to have photosynthetic characteristics intermediate between C3 and C4 plants6–8. We now report that a single plant of Mollugo nudicaulis can have some leaves with C3 characteristics and others with C4 characteristics according to their position on the stem.

Inhibition of isocitrate lyase: the basis for inhibition of growth of two Arthrobacter species by pyruvate.

Growth of Arthrobacter atrocyaneus and A. pyridinolis on certain growth substrates was found to be inhibited by pyruvate and compounds which can be converted to pyruvate. Growth of A. atrocyaneus on acetate, for example, was completely inhibited by 5 mm pyruvate; growth of this organism on glucose was less sensitive and growth on succinate was insensitive to inhibition by pyruvate. Growth of a third Arthrobacter species, A. crystallopoietes, on acetate and other substrates was not inhibited by pyruvate. The site of pyruvate inhibition was shown to be the isocitrate lyase reaction. Glyoxylate, which affords a bypass of this reaction, restored the ability of A. atrocyaneus to evolve (14)CO(2) from acetate in the presence of pyruvate. The isocitrate lyases from A. atrocyaneus and A. pyridinolis were competitively inhibited by concentrations of pyruvate as low as 1 mm, whereas the enzyme from A. crystallopoietes was unaffected by this concentration of pyruvate. Comparable levels of phosphoenolpyruvate did not inhibit the isocitrate lyases from any of the species. A mutant strain of A. atrocyaneus, PW11, which is deficient in isocitrate lyase activity, grew on glucose at a reduced rate that was comparable to the rate of growth of the wild-type strain on glucose plus lactate. Addition of lactate to PW11 did not further reduce its rate of growth on glucose. Thus, the glyoxylate pathway appears to be used as an anaplerotic pathway during growth of A. atrocyaneus on glucose. Two other considerations suggest that A. atrocyaneus and A. pyridinolis, but not A. crystallopoietes, may be deficient in the ability to convert pyruvate to 4-carbon acids. First, the former two species accumulate intracellular pyruvate from exogenous l-alanine to a much greater extent than does A. crystallopoietes. Moreover, A. atrocyaneus and A. pyridinolis are incapable of growth on lactate as sole source of carbon whereas A. crystallopoietes can grow on lactate.

C-4 and C-3 Carboxylation Characteristics in the Genus Zygophyllum (Zygophyllaceae)

C-4 and C-3 Carboxylation Characteristics in the Genus Zygophyllum (Zygophyllaceae)

An estimation of pyruvate recycling during gluconeogenesis in the perfused rat liver

To estimate the degree of recycling of pyruvate during gluconeogenesis, an isotope tracer procedure was employed. Using the isolated, perfused rat liver with pyruvate-2- 14C in the perfusion fluid, the 3-carbon acids lactate and pyruvate were isolated and the distribution of 14C in each carbon was assayed. It can be shown that the degree of recycling can be approximated as twice the sum of 14C in carbons 1 and 3. Glucose, acetoacetate, and β-hydroxybutyrate were also determined, and their 14C distribution estimated by appropriate degradation procedures. In livers from fasted rats, recycling of pyruvate during 1 hr incubation occurred at a rate of 0.21 μmoles ± 0.02 (SE)/min/g while gluconeogenesis occurred at a rate of 0.49 ± 0.11 μmoles pyruvate-2- 14C/min/g. In livers from carbohydrate-fed rats, the ratio was reversed, with 0.35 ± 0.06 μmoles pyruvate-2- 14C recycled and only 0.09 ± 0.03 μmoles converted to glucose. These patterns were not affected by the simultaneous presence of octanoate in the perfusion, during which ketone body production was greatly increased. Only about 20% of the ketone bodies formed were derived from pyruvate, much less with octanoate present, and over 95% of the total radioactivity was in carbons 1 and 3 of acetoacetate as anticipated from the degree of pyruvate recycling. The glucose invariably had 3–4% of its total activity in carbons 3 and 4 and the remainder distributed approximately equally in carbons 1, 2, 5, and 6. The radioactivity in respired CO 2 indicated that about 13–25% of the total O 2 uptake was due to pyruvate oxidation to CO 2. The data indicate that during the course of liver perfusion a part of the pyruvate substrate is recycled back to pyruvate after conversion to 4-carbon acids. A major portion is recycled directly via the dicarboxylic acid cycle while a minor portion is recycled after traversing the tricarboxylic acid cycle. The extent of recycling was increased greatly by carbohydrate feeding while conversion to glucose was greatly reduced. Recycling may occur via three mechanisms: reversal of pyruvate carboxylase, the malic enzyme, and pyruvate kinase, but it is assumed that, the major path is via pyruvate kinase; and the results point to the possibility that the activity of this enzyme may regulate the rate of glucose production.

Compartments of organic acids in the synthesis of asparagine and homoserine in pea roots

14C-Labeled aspartate and organic acids were supplied to pea-root pieces to elucidate the pathways of carbon leading to asparagine and homoserine synthesis. The pattern of labeling in the products, and the effects of competing non-radioactive acids on the distribution of 14C from labeled substrates, gave the following results. Supplied aspartate is not converted directly to asparagine or homoserine, but must first enter the Krebs cycle. If aspartate is an immediate precursor of either of these compounds, the synthetic pathways must be compartmented from externally supplied aspartate. Carbon leaves the Krebs cycle as succinate, and is converted to other 4-carbon acids in a metabolic system that is separated from the Krebs cycle and presumably outside the mitochondria. The pathway from succinate to homoserine proceeds via extramitochondrial fumarate and malate, and to asparagine via fumarate. Carboxylation of pyruvate (or a related three-carbon acid) leads to homoserine and asparagine via a symmetrical intermediate permitting equilibration of the two ends of the 4-carbon precursor. About four times more carbon from added pyruvate is converted to homoserine and asparagine via the Krebs cycle than by carboxylation.

The distribution and properties of alkaline inorganic pyrophosphatase from higher plants

An alkaline inorganic pyrophosphatase (E.C. 3.6.1.1) has been detected in a range of species and is associated at least partly with the chloroplast fraction. It is as active in plants showing only the carbon reduction cycle of carbon dioxide fixation as in plants exhibiting the 4-carbon acid pathway. The enzyme has been isolated and partially purified from sugar cane leaves. It has an absolute requirement for magnesium ions and shows a pH optimum of 8·3. It is inhibited by orthophosphate, arsenate and EDTA but is unaffected by -SH group inhibitors. Possible functions of the pyrophosphatase are discussed.

THE EFFECT OF AUXINS ON PROTOPLASMIC STREAMING. III

1. A new method is described which gives a continuous record of the absolute rate of protoplasmic streaming in epidermal cells of the Avena coleoptile. 2. With this method a study was made of the influence of malate and iodoacetate on streaming velocity, in order to make correlations with the previously established effects of these substances on growth and respiration. 3. In the presence of optimum concentrations of indole-3-acetic acid in freshly cut sections, malate had no effect on streaming. In the presence of very low concentrations of the auxin, however, malate increased the range of response, so that the threshold of auxin sensitivity was lowered some ten times by the malate. Malate alone had no effect on streaming. 4. In coleoptile sections, soaked overnight in sugar solution or in water, the acceleration of streaming normally caused by auxin almost disappears, but the presence of malate causes large accelerations of streaming by the auxin. 5. Similarly, in sections from old coleoptiles, which no longer show acceleration of streaming by auxin, the acceleration is restored when malate is added together with the auxin. 6. Malate does not enter the cell as rapidly as does auxin, but easily detectable amounts penetrate within 30 minutes. 7. Iodoacetate in the concentration which inhibits growth (5 x 10(-5)M) completely inhibits the acceleration of streaming by auxin. In still lower concentrations iodoacetate slightly accelerates streaming. Higher concentrations, up to 2 x 10(-4)M, did not reduce the rate of streaming below that of controls without auxin. The effect of iodoacetate is therefore to inhibit the acceleration caused by auxin and not to affect the basal streaming rate. 8. It is concluded that, just as for growth and respiration, malate is necessary for the response to auxin shown by acceleration of streaming. This further strengthens the triple parallel between the effects of auxin on streaming, growth, and respiration, all of which are apparently mediated by the 4-carbon acid system.

THE RELATION BETWEEN THE FOUR-CARBON ACIDS AND THE GROWTH OF OAT SEEDLINGS

Plant growth hormones are noted for the multiplicity of their effects. This is particularly true of the auxins, which are known to influence elongation of roots and shoots, determination of root number, production of callus tissue, tropistic responses, correlative growth of plant organs and protoplasmic streaming. The most striking feature about the variety of these auxin effects is the fact that any one concentration of the hormone will often stimulate one process, inhibit another, and have no effect on a third. If the concentration is varied these relations may be completely altered. Such effects, together with the fact that the auxins have an effect on protoplasmic streaming, have suggested that these hormones must play a decisive r6ole in some fundamental and common intracellular process. For some time attempts had been made to demonstrate a link between auxin and cell respiration. Experiments showed, however, that auxin had no demonstrable effect on the respiratory rate of plant cells (Bonner, 1936; Van Hulssen, 1936) and the conclusion was drawn that the hormone had no respiratory effect (DuBuy, 1940). More recently, however, Commoner and Thimann (1941) have shown that indole-3-acetic acid is directly related to the activity of a specific respiratory process, the 4-carbon dicarboxylic acid system. They showed that the salts of these acids (malic, fumaric, and succinic) will enhance the effect of auxin on growth and that the inhibitory effect of iodoacetic acid on growth is due to a specific inhibition of this system. It was shown that the 4-carbon acid system normally accounted for only 5-10 per cent of the total respiration, but that all the growth is dependent upon its activity. Thus it was demonstrated that auxin can stimulate the respiratory activity of this system (and in this way increase the rate of oxygen consumption) and that the effect of the 4-carbon acids on respiration is enhanced by the presence of auxin. This work dealt with a single effect of auxin, the elongation of ex314

Oxidation of Reduced Codehydrogenase I

THE discovery of an enzyme1,2 capable of catalysing the oxidation of reduced codehydrogenase I (cozymase, CoI) at an extremely rapid rate3 naturally raises the question as to what part this enzyme plays in hydrogen transport. There is considerable evidence1,2,4,5 that this enzyme (diaphorase) catalyzes the reaction between dihydrocozymase and the cytochrome system, and Dewan and Green2 have suggested that cytochromes A and B (but not C) and cytochrome oxidase are involved. The method of preparing diaphorase is such that succinic acid dehydrogenase is also present in large amounts. With the possible exception of the recent announcement3 that diaphorase had been obtained in highly purified form, it seems fairly certain that most of the data in the literature regarding either enzyme were obtained with preparations containing both enzymes. This statement has a very important bearing on the 4-carbon acid catalysis theory of Szent-Gyorgyi, since it raises the question as to the possibility that succinic acid dehydrogenase is an intermediary hydrogen carrier between diaphorase and cytochrome.