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Phosphoglucoisomerase-catalysed interconversion of hexose phosphates. Kinetic study by 13C n.m.r. of the phosphoglucoisomerase reaction in 2H2O.

ABSTRACT: The fate of D-[2-13C]glucose 6-phosphate exposed to phosphoglucoisomerase (glucose-6-phosphate isomerase, EC 5.3.1.9) in 2H2O was monitored by 13C-n.m.r. spectroscopy. The generation of the anomers of both D-[2-13C]fructose 6-phosphate and D-[2-13C,2-2H]glucose 6-phosphate followed a single-exponential pattern. The rate constant, which was proportional to the enzyme concentration, was about 14 times higher, however, in the former than in the latter case. The disappearance of D-[2-13C,2-1H]glucose 6-phosphate occurred in a bi-exponential manner, the rate constants for the fast and the slow processes being in fair agreement with those obtained for the generation of D-[2-13C]fructose 6-phosphate and D-[2-13C,2-2H]glucose 6-phosphate respectively. These findings indicate that the process of equilibration of D-[2-13C]glucose 6-phosphate and D-[2-13C]fructose 6-phosphate is at least one order of magnitude faster than the intermolecular proton transfer involving the deuterons from the solvent. Such a difference provides strong support to the view that the inverconversion of hexose phosphates in the reaction catalysed by phosphoglucoisomerase proceeds in two distinct steps, the second of which occurs according to two competing modalities with either an intramolecular or an intermolecular proton transfer.

SUBMITTER: Willem R

PROVIDER: S-EPMC1136914 | biostudies-other | 1990 Jan

REPOSITORIES: biostudies-other

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Project description:Kinetic models of metabolic networks offer the promise of quantitative phenotype prediction. The mechanistic characterization of enzyme catalyzed reactions allows for tracing the effect of perturbations in metabolite concentrations and reaction fluxes in response to genetic and environmental perturbation that are beyond the scope of stoichiometric models. In this study, we develop a two-step computational pipeline for the rapid parameterization of kinetic models of metabolic networks using a curated metabolic model and available 13C-labeling distributions under multiple genetic and environmental perturbations. The first step involves the elucidation of all intracellular fluxes in a core model of E. coli containing 74 reactions and 61 metabolites using 13C-Metabolic Flux Analysis (13C-MFA). Here, fluxes corresponding to the mid-exponential growth phase are elucidated for seven single gene deletion mutants from upper glycolysis, pentose phosphate pathway and the Entner-Doudoroff pathway. The computed flux ranges are then used to parameterize the same (i.e., k-ecoli74) core kinetic model for E. coli with 55 substrate-level regulations using the newly developed K-FIT parameterization algorithm. The K-FIT algorithm employs a combination of equation decomposition and iterative solution techniques to evaluate steady-state fluxes in response to genetic perturbations. k-ecoli74 predicted 86% of flux values for strains used during fitting within a single standard deviation of 13C-MFA estimated values. By performing both tasks using the same network, errors associated with lack of congruity between the two networks are avoided, allowing for seamless integration of data with model building. Product yield predictions and comparison with previously developed kinetic models indicate shifts in flux ranges and the presence or absence of mutant strains delivering flux towards pathways of interest from training data significantly impact predictive capabilities. Using this workflow, the impact of completeness of fluxomic datasets and the importance of specific genetic perturbations on uncertainties in kinetic parameter estimation are evaluated.

Dataset Information

Phosphoglucoisomerase-catalysed interconversion of hexose phosphates. Kinetic study by 13C n.m.r. of the phosphoglucoisomerase reaction in 2H2O.

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