Effect of reversible reactions on isotope label redistribution

Abstract

The pentose phosphate pathway plays several key roles in metabolism including supply of biosynthetic carbon skeletons and reducing power. Previous research has focused on determining the fluxes through the reactions of this pathway using carbon-labeled substrates and models that make certain assumptions about the reversibility of the transketolase and transaldolase reactions in the nonoxidative pathway. These assumptions, however, have resulted in inconsistencies between the predicted carbon label distributions using these models and those determined experimentally. A general metabolic reaction network model developed in this paper and applied to the pentose phosphate pathway not only incorporates reaction reversibility but also accounts for the effect of individually varying extents of reaction reversibility on labeled carbon fractional enrichment values for intermediate metabolites. In addition, an algorithm is presented that can be used to calculate the three individual transaldolase and transketolase extents of reversibility. The results of this method show that varying extents of reaction reversibility have an observable effect on the metabolite carbon label distributions which can in turn affect flux calculation for other parts of the metabolic network such as the tricarboxylic acid cycle. In addition, the observability of reversibility extent and accuracy of flux calculations depend on the particular choice of metabolite carbon enrichments measured. In particular, [6-13 C]hexose 6-phosphate and [4-13 C]erythrose 4-phosphate carbon enrichment values resulting from [1- 13C]glucose feeding contained more information as compared to those from ribose 5-phosphate. This analysis was applied to literature data of metabolite carbon labeling that resulted from supplying either 13 C-or 14 C-enriched substrates to several cell types growing under various conditions. The specific activities of metabolite carbon atoms taken from rat epididymal adipose tissue, goosefish islet cells, Corynebacterium glutamicum, and Escherichia coli supplied with either [2-14 C]glucose or [1-13 C]glucose demonstrate how reversibility is present in the pentose phosphate pathway and the extents of reversibility can be estimated from labeled carbon data sets.Keywords : pentose-phosphate pathway ; metabolic flux ; isotope-exchange reaction; mathematical modeling;13 C enrichment.An important parameter in metabolic pathway analysis is the Mancuso et al., 1994;Marx et al., 1996 ; Park et al., 1997;Sharfstein et al., 1994; Vallino and Stephanopoulos, 1993, flux of carbon through the pathway. Accurate determination of metabolic fluxes in vivo is a critical step in elucidating flux con-1994a, b; Wiechert et al., 1997a, b ;Zupke and Stephanopoulos, 1995). Isotope labeling techniques are particularly useful due to trol in metabolic networks. Sensitive experimental techniques the additional information contained in the metabolite carbon and rigorous modeling methods are both required for the calculabel fractional enrichment values resulting from the s...

A Flux Model of Glycolysis and the Oxidative Pentosephosphate Pathway in Developing Brassica napus Embryos

“…In the network formed by glycolysis and OPPP, cyclic fluxes and reversible reactions cause the redistribution of label among different intermediates in ways that are not easily understood by inspection of labeling patterns (30,65). Computer-aided modeling is needed if 13 C label at multiple carbon positions is to be quantitatively interpreted (37,66).…”

The pathogenesis of transaldolase deficiency

“…In addition, reduced levels of TAL result in increased G6PD and 6PGD activities, and increased GSH levels, with inhibition of apoptosis. The impact of TAL on apoptotic signaling (23) may be related to an overwhelming influence of TALcatalyzed dihydroxyacetone transfer reactions on the distribution of flux between the PPP and nucleotide metabolism which determines overall propagation of biochemical signals in the context of a metabolic network (43,65). TAL activity has a dominant influence on the balance between the two branches of PPP (23) and, therefore controls intracellular NADPH levels and neutralization of ROIs (43).…”

Efficient modeling of 13C-labeling distributions in microorganisms

“…This reduction is often applied to the hexose 6-phosphate pools (g6p and f6p) in the glycolysis and the pentose 5-phosphate pools (ru5p, x5p, r5p) in the pentose phosphate pathway (e.g. Schmidt et al, 1997;Follstad and Stephanopoulos, 1998). Lumping g6p and f6p and lumping ru5p, x5p and r5p further simplifies the metabolic reaction network of Fig.2 to the reduced version shown in Fig.3.…”

“…Bondomers of a given compound are denoted by an abbreviation of the compound and a binary subscript. Whereas in isotopomer notation the binary subscript '0' denotes a 12 C-atom and a '1' a 13 C-atom (Schmidt et al, 1997), in bondomer notation '0' denotes a C-C bond that has been formed in one of the metabolic reactions and '1' denotes a C-C bond that was already present in the medium substrate molecule. A linear or branched molecule that has a backbone of n carbon atoms has n-1 C-C bonds.…”

“…Metabolic reaction rates are subsequently determined by fitting the labeling measurements with a model of the metabolic reaction network wherein the reaction rates are the free variables. A mathematical model for this system traditionally consists of a set of balances for all isotopomers of each metabolite in the reaction network (Schmidt et al, 1997). Isotopomers are chemically identical molecules that only vary with respect to their 12 C and 13 C-distribution.…”