Marion H. O'Leary

Carbon isotope fractionation in plants

Abstract Plants with the C 3 , C 4 , and crassulacean acid metabolism (CAM) photosynthetic pathways show characteristically different discriminations against 13 C during photosynthesis. For each photosynthetic type, no more than slight variations are observed within or among species. CAM plants show large variations in isotope fractionation with temperature, but other plants do not. Different plant organs, subcellular fractions and metabolises can show widely varying isotopic compositions. The isotopic composition of respired carbon is often different from that of plant carbon, but it is not currently possible to describe this effect in detail. The principal components which will affect the overall isotope discrimination during photosynthesis are diffusion of CO 2 , interconversion of CO 2 and HCO − 3 , incorporation of CO 2 by phosphoenolpyruvate carboxylase or ribulose bisphosphate carboxylase, and respiration. Theisotope fractionations associated with these processes are summarized. Mathematical models are presented which permit prediction of the overall isotope discrimination in terms of these components. These models also permit a correlation of isotope fractionations with internal CO 2 concentrations. Analysis of existing data in terms of these models reveals that CO 2 incorporation in C 3 plants is limited principally by ribulose bisphosphate carboxylase, but CO 2 diffusion also contributes. In C 4 plants, carbon fixation is principally limited by the rate of CO 2 diffusion into the leaf. There is probably a small fractionation in C 4 plants due to ribulose bisphosphate carboxylase.

Biochemical basis of carbon isotope fractionation.

Publisher Summary This chapter provides an overview of the physical and chemical components that give rise to isotope fractionation in plants. The chapter discusses as to how these components are put together to form integrated theories of plant isotope fractionation. Plants fractionate carbon isotopes during photosynthesis. The magnitude of the fractionation varies with photosynthetic type, environment, genotype, and other factors, and this variation can be used to study various issues in plant physiology. About 1.1% of all carbon atoms in natural materials are the nonradioactive isotope carbon-13. Precise measurements of the 13C content of CO2 are carried out using an isotope ratio mass spectrometer. The chapter discusses the components of isotope fractionation in plants. Isotope fractionation in plants is best understood by beginning with the individual physical and chemical processes that contribute to the overall isotope fractionation. Isotope fractionations may be of two types: (1) thermodynamic and (2) kinetic. Thermodynamic fractionations reflect differences in equilibrium constants for isotopic species. Kinetic fractionations reflect differences in rate constants for isotopic species.

Spatial and temporal variation in carbon isotope discrimination in prairie graminoids

We present the results of a 5-year examination of variation in the stable carbon isotope composition (δ) of three C3 graminoid species from a Sandhills prairie: Agropyron smithii, Carex heliophila and Stipa comata. Although consistent species-specific patterns for mean δ were seen, there was also significant and substantial among-year and within-season variation in δ. A smaller contribution to variation in δ came from topographic variation among sampling sites. Effects of species, year, season and topography contribute to variation in δ in an additive manner. An association between climate and δ exists that is consistent with previous work suggesting that δ reflects the interplay between photosynthetic gas exchange and plant water relations. Within the growing season, the time over which δ integrates plant response to the environment ranges from days to months.

Equilibrium carbon isotope effect on a decarboxylation reaction

Abstract Carbon isotope exchange equilibrium between carbon dioxide and the β-carboxyl carbon of D-isocitric acid has been achieved by means of the enzyme isocitrate dehydrogenase. Measurements of the isotopic compositions at these two sites reveal that the carboxyl carbon is enriched in 13C relative to carbon dioxide. The isotope exchange equilibrium constant is 1.0027 at 25°, pH 7.5.

Regulation of malic-acid metabolism in Crassulacean-acid-metabolism plants in the dark and light: In-vivo evidence from 13C-labeling patterns after 13CO2 fixation

The labeling patterns in malic acid from dark 13CO2 fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and 13C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12–14 h dark 13CO2 fixation the ratio of [4-13C] to [1-13C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The 13C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87–89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following 13CO2 fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [13C] malic acid (13CO2 or [1-13C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to 13CO2 in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284–291). In the shortest times, only singly-labeled [4-13C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.

Carbon isotope effect on the enzymatic decarboxylation of pyruvic acid

Abstract The decarboxylation of pyruvic acid by the thiamine pyrophosphate dependent pyruvate decarboxylase from brewers yeast is accompanied by a carboxyl carbon isotope effect k 12 k 13 = 1.0083±0.0003 at 25°, pH 6.8. The small size of the isotope effect indicates that decarboxylation is not rate-determining in the overall reaction. The rate constant for decarboxylation of the enzyme-bound pyruvate-thiamine pyrophosphate complex is greater by about a factor of five than the rate constant for dissociation of this complex to form free pyruvate and the enzyme-thiamine pyrophosphate complex.

Arginine as a substrate binding site in aspartate aminotransferase

Abstract Modification of one or two arginine residues in pig-heart cytoplasmic aspartate aminotransferase with 1,2-cyclohexanedione nearly abolishes its catalytic activity and abolishes its ability to bind dicarboxylic acids. The modification is competitively inhibited by glutaric acid. Modification of the enzyme causes no change in its ability to transaminate alanine, but causes a tenfold increase in the Michaelis constant and a 10 4 fold decrease in the rate of transamination of aspartate. These results indicate that the binding site for the β-carboxyl group of aspartic acid is an arginine residue.

A proposed structure for the 330 -nm chromophore of glutamate decarboxylase and other pyridoxal 5'-phosphate dependent enzymes.

Abstract 1. 1. Pyridoxal 5′-phosphate reacts with 1,3-diaminopropane to form a cyclic aldamine structure, rather than a Schiff base. The structure of this compound was proved by direct spectral study and by analogy with the reaction of the coenzyme with n -butylamine and the reactions of pyridine-4-aldehyde with n -butylamine and with 1,3-diaminopropane. 2. 2. Evidence is presented which suggests that the 330 nm chromophore of glutamate decarboxylase is an aldamine formed by addition of a second amino group to the carbon nitrogen double bond, rather than any other sort of aldamine or a normal Schiff base structure.

6 Catalytic Strategies in Enzymic Carboxylation and Decarboxylation

Publisher Summary Living things absorb CO 2 from the atmosphere by photosynthesis and return it to the atmosphere by respiration. The biochemical reactions associated with these processes are the subject of this chapter. This chapter discusses the way in which enzymes catalyze carboxylations and decarboxylations. The first concern is the chemical mechanism of the reaction, that is, a description of the intermediates that occur along the reaction path and the chemical transformations that connect these intermediates. Second is the mechanistic strategy of the reaction—that is, a description of the underlying catalytic forces and effects used by the enzyme to achieve the rate acceleration and specificity observed. For most carboxylases and decarboxylases, the chemical mechanisms are relatively well understood. On the other hand, mechanistic strategies are much more elusive, depending as they do on a detailed knowledge of enzyme structure and action. There is a basic unity of mechanistic strategies for carboxylations and decarboxylations that can equally well be applied to number of cases.

1-Hydroxycyclopropane carboxylic acid phosphate: A potent inhibitor of enzymes metabolizing phosphoenolpyruvate

Abstract 1-Hydroxycyclopropane carboxylic acid phosphate has been synthesized from diethyl succinate by acyloin condensation followed by ring contraction and phosphorylation. This compound is a potent competitive inhibitor of enzymes utilizing phosphoenolpyruvate. For phosphoenolpyruvate from maize, Ki = 7.3 μM at pH 8.0 in the presence of Mg2+. For pyruvate kinase, Ki = 2.0 mM at pH 7.0. For enolase, Ki = 8.0 μM at pH 8.0. In each case, this compound is a substantially better inhibitor than the commonly used phosphoenolpyruvate analogs phosphoglycolate and phospholactate, presumably because of the similarity in geometric and electronic structure between the cyclopropane compound and phosphoenolpyruvate.