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On the Role of Catabolic Enzymes in Biosynthetic Models of Glycogen Molecular Weight Distributions.

ABSTRACT: Glycogen and starch are complex branched polymers of glucose that serve as units of glucose storage in animals and plants, respectively. Changes in the structure of these molecules have been linked to changes in their respective functional properties. Enzymatic models of starch synthesis have provided valuable insights into the biosynthetic origins of starch structure and functional properties but have not successfully been applied to glycogen despite the structural similarities between the two polymers. Modifications to biosynthetic models of starch structure were tested for applicability to glycogen. Mathematical evidence is provided showing the necessity (which has hitherto been in doubt) of considering the effects of catabolic (degradative) enzymes in biosynthesis-based approaches that seek to accurately describe the molecular weight distributions of individual chains of glycogen formed in vivo through glycogenesis. This finding also provides future direction for inferring the dependence of enzyme activities on substrate chain length from in vivo data.

SUBMITTER: Nada SS

PROVIDER: S-EPMC6641748 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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On the Role of Catabolic Enzymes in Biosynthetic Models of Glycogen Molecular Weight Distributions.

Nada Sharif S SS Gilbert Robert G RG

ACS omega 20170831 8

Glycogen and starch are complex branched polymers of glucose that serve as units of glucose storage in animals and plants, respectively. Changes in the structure of these molecules have been linked to changes in their respective functional properties. Enzymatic models of starch synthesis have provided valuable insights into the biosynthetic origins of starch structure and functional properties but have not successfully been applied to glycogen despite the structural similarities between the two ...[more]

PMID: 31457793

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Project description:IntroductionRice is a primary global food source, and its production is affected by abiotic stress, caused by climate change and other factors. Recently, the pyrimidine reductive catabolic pathway, catalyzed by dihydropyrimidine dehydrogenase (DHPD), dihydropyrimidinase (DHP) and β-ureidopropionase (β-UP), has emerged as a potential participant in the abiotic stress response of rice.MethodsThe rice enzymes were produced as recombinant proteins, and two were kinetically characterized. Rice dihydroorotate dehydrogenase (DHODH), an enzyme of pyrimidine biosynthesis often confused with DHPD, was also characterized. Salt-sensitive and salt-resistant rice seedlings were subjected to salt stress (24 h) and metabolites in leaves were determined by mass spectrometry.ResultsThe OsDHPD sequence was homologous to the C-terminal half of mammalian DHPD, conserving FMN and uracil binding sites, but lacked sites for Fe/S clusters, FAD, and NADPH. OsDHPD, truncated to eliminate the chloroplast targeting peptide, was soluble, but inactive. Database searches for polypeptides homologous to the N-terminal half of mammalian DHPD, that could act as co-reductants, were unsuccessful. OsDHODH exhibited kinetic parameters similar to those of other plant DHODHs. OsDHP, truncated to remove a signal sequence, exhibited a kcat/Km = 3.6 x 103 s-1M-1. Osb-UP exhibited a kcat/Km = 1.8 x 104 s-1M-1. Short-term salt exposure caused insignificant differences in the levels of the ureide intermediates dihydrouracil and ureidopropionate in leaves of salt-sensitive and salt-resistant plants. Allantoin, a ureide metabolite of purine catabolism, was found to be significantly higher in the resistant cultivar compared to one of the sensitive cultivars.DiscussionOsDHP, the first plant enzyme to be characterized, showed low kinetic efficiency, but its activity may have been affected by truncation. Osb-UP exhibited kinetic parameters in the range of enzymes of secondary metabolism. Levels of two pathway metabolites were similar in sensitive and resistant cultivars and appeared to be unaffected by short-term salt exposure."

Project description:BackgroundStable isotope tracers are used to assess metabolic flux profiles in living cells. The existing methods of measurement average out the isotopic isomer distribution in metabolites throughout the cell, whereas the knowledge of compartmental organization of analyzed pathways is crucial for the evaluation of true fluxes. That is why we accepted a challenge to create a software tool that allows deciphering the compartmentation of metabolites based on the analysis of average isotopic isomer distribution.ResultsThe software Isodyn, which simulates the dynamics of isotopic isomer distribution in central metabolic pathways, was supplemented by algorithms facilitating the transition between various analyzed metabolic schemes, and by the tools for model discrimination. It simulated 13C isotope distributions in glucose, lactate, glutamate and glycogen, measured by mass spectrometry after incubation of hepatocytes in the presence of only labeled glucose or glucose and lactate together (with label either in glucose or lactate). The simulations assumed either a single intracellular hexose phosphate pool, or also channeling of hexose phosphates resulting in a different isotopic composition of glycogen. Model discrimination test was applied to check the consistency of both models with experimental data. Metabolic flux profiles, evaluated with the accepted model that assumes channeling, revealed the range of changes in metabolic fluxes in liver cells.ConclusionsThe analysis of compartmentation of metabolic networks based on the measured 13C distribution was included in Isodyn as a routine procedure. The advantage of this implementation is that, being a part of evaluation of metabolic fluxes, it does not require additional experiments to study metabolic compartmentation. The analysis of experimental data revealed that the distribution of measured 13C-labeled glucose metabolites is inconsistent with the idea of perfect mixing of hexose phosphates in cytosol. In contrast, the observed distribution indicates the presence of a separate pool of hexose phosphates that is channeled towards glycogen synthesis.

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On the Role of Catabolic Enzymes in Biosynthetic Models of Glycogen Molecular Weight Distributions.

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On the Role of Catabolic Enzymes in Biosynthetic Models of Glycogen Molecular Weight Distributions.

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