Time course of key short-chain reaction rates and regulation of the MCKAT-C4 reaction.

<p><b>(A)</b> Time course of the reaction rates of MCKAT for its C6 and C4 substrates (vMCKATC6 and vMCKATC4, respectively) and of the summed activities of SCAD and MCAD on their C4 substrate (vMCADC4 + vSCADC4) after a sudden upshift of [palmitoyl-CoA]_CYT from 0.1 to 60 μM. <b>(B)</b> Time course of regulatory metabolite contributions to vMCKATC4 after a sudden [palmitoyl-CoA]_CYT increase from 0.1 to 60 μM.</p

Regulation of key reactions by their substrates and products.

<p><b>(A-F)</b> Relative average absolute contribution, i.e. , of metabolites to the transition from the steady state at 0.1 μM to the indicated concentration of palmitoyl-CoA, calculated for vMCKATC6 (panel A), vMCKATC4 (panel B), vMCADC6 (panel C), vSCADC4 (panel D), vMPTC8 (panel E), vCPT2C16 (panel F).</p

Predictions from the model of yeast glycolysis compared to experimental data from [23].

<p> Black bars represent the experimental (± SEM) data and white bars represent model predictions. The <i>K_i</i> of HXK for T6P was 0.2 mM for the non-starved cells from the respirofermentative culture (D = 0.35 h⁻¹) and 0.04 mM for the other three conditions. Abbreviations as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002483#pcbi-1002483-g001" target="_blank">Figure 1</a>.</p

The glycolytic and fermentative pathway as they were modeled in this study.

<p>Metabolites are depicted in bold face, allosteric regulators in regular, enzymes in italics and branching pathways underlined. GLCo: extracellular glucose, GLCi: intracellular glucose, G6P: glucose 6-phosphate, F6P: fructose 6-phosphate, F16BP: fructose 1,6-bisphosphate, DHAP: dihydroxyacetone phosphate, GAP: glyceraldehyde 3-phosphate, BPG: 1,3-bisphosphoglycerate, 3PG: 3-phosphoglycerate, 2PG: 2-phosphoglycerate, PEP: phosphoenolpyruvate, PYR: pyruvate, ACE: acetaldehyde, EtOH: ethanol, HXT: hexose transport, HXK: hexokinase (EC 2.7.1.1), PGI: phosphoglucose isomerase (EC 5.3.1.9), PFK: phosphofructokinase (EC 2.7.1.11), ALD: aldolase (EC 4.1.2.13), TPI: triose-phosphate isomerase (EC 5.3.1.1), GAPDH: glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), PGK: 3-phosphoglycerate kinase (EC 2.7.2.3), GPM: phosphoglycerate mutase (EC 5.4.2.1), ENO: enolase (EC 4.2.1.11), PYK: pyruvate kinase (EC 2.7.1.40), PDC: pyruvate decarboxylase (EC 4.1.1.1), ADH: alcohol dehydrogenase (EC 1.1.1.1).</p

Elucidating the mechanism of flux decline.

<p><b>1.</b> Upon substrate overload with cytosolic palmitoyl-CoA, MCKAT’s ketoacyl-CoA substrates start to accumulate. <b>2.</b> The unfavourable equilibrium constant of the preceding enzyme, medium/short-chain hydroxyacyl-CoA dehydrogenase, works as an amplifier, leading to the accumulation of upstream CoA esters, including acyl-CoA esters. <b>3.</b> These acyl-CoA esters are at the same time products of MCKAT and inhibit its already low activity further. <b>4.</b> Finally, the accumulation of CoA esters leads to a sequestration of free CoA. CoA being a cofactor for MCKAT, its sequestration limited the MCKAT activity even further, thus completing the vicious cycle. <b>5</b>. Since CoA is also a substrate for distant enzymes (such as MTP), it efficiently communicates the ‘traffic jam’ at MCKAT to the entire pathway.</p

Simulated steady-state fluxes and concentrations in the mFAO model.

<p><b>(A)</b> The effect of cytosolic palmitoyl-CoA concentration ([palmitoyl-CoA]_CYT) on the steady-state flux. The steady-state uptake flux of palmitoyl-CoA (J_uptake) is plotted (calculated as the steady-state flux of palmitoyl-carnitine through CACT, i.e. the uptake of palmitoyl-carnitine into the mitochondria), in contrast to van Eunen <i>et al</i>. 2013 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005461#pcbi.1005461.ref008" target="_blank">8</a>] in which the NADH flux was plotted. The two are uniquely linked with the latter being 7-fold higher. <b>(B)</b> The steady-state concentrations of free CoA (identical to data from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005461#pcbi.1005461.ref008" target="_blank">8</a>]), the sum of all intermediate CoA esters, the sum of all C4- and C6 CoA esters and the subset of C4- and C6 CoA esters (new results). <b>(C-D)</b> Distribution of steady-state fluxes (J) of different chain-length substrates through MTP and MCKAT, respectively. Only the chain lengths which are converted by these enzymes (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005461#pcbi.1005461.g001" target="_blank">Fig 1</a>) are included.</p

The role of the short-chain branch.

<p><b>(A)</b> Steady-state flux versus [palmitoyl-CoA]_CYT in the published model (black line), a model without any enzymatic promiscuity (black dashed line, reproducing results from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005461#pcbi.1005461.ref008" target="_blank">8</a>] as a reference), a model without promiscuity of MCKAT (green line), and finally a model with only promiscuity of MCKAT, but not of any of the other enzymes (purple line). <b>(B)</b> The flux control coefficients in the original model as a function of [palmitoyl-CoA]_CYT. Only enzymes with a substantial contribution are included; for a complete set of flux control coefficients, see figure A in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005461#pcbi.1005461.s002" target="_blank">S2 Fig</a>. <b>(C-D)</b> Top 15 flux response coefficients with the highest absolute values at 25 μM (C) and 60 μM (D) of [palmitoyl-CoA]_CYT. The parameters <i>p</i> for which the flux response coefficient was calculated is indicated on the Y-axis. Dark grey bars represent CPT1-related parameters, pink bars represent M/SCHAD-related parameters, purple bars represent MCKAT-related parameters and the light blue bar represents a crotonase-related parameter. <b>(E)</b> Flux response coefficient of [NAD⁺]/[NADH] () partitioned in contributions of individual NAD⁺-dependent reactions at 5 palmitoyl-CoA concentrations according to . (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005461#pcbi.1005461.e009" target="_blank">Eq 4</a>). The contributions of M/SCHAD reactions C8-C16 to were negligible and therefore not shown in the legend.</p

Simulation of a sudden upshift of the extracellular glucose concentration starting from a steady-state, aerobic, glucose-limited chemostat culture at a dilution rate of 0.1 h⁻¹.

<p>The ATP concentration was decreased by 50% at the onset of the glucose upshift, maintaining it constant thereafter. Experimental data are taken from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002483#pcbi.1002483-Visser3" target="_blank">[26]</a> with permission. For details of the simulation, see text. A complete list of parameters values used is given in Table S6 of the supporting information. All other parameters were kept the same as in the non‐starved cells from the respiratory culture (D=0.1 h⁻¹).</p

The comparison of the model results of yeast glycolysis obtained with the original model of Teusink <i>et al. </i>[<b>12</b>] (panel A–D), the model developed in this study (panel E–L) and the latter model without activation of pyruvate kinase by fructose 1,6-bisphosphate (panel M–P; see text for model description).

<p>The data used in these simulations were from the non-starved cells of the glucose-limited respiratory culture (D = 0.1 h⁻¹). The <i>V_max</i> values were measured in either the <i>in vivo</i>-like assay medium (panel A–D and I–P) or in the assay medium optimized for each enzyme (panel E–H). In the former case also the other glyceraldehyde-3-phosphate dehydrogenase parameters measured under <i>in vivo</i>-like conditions were used; in the latter case the original glyceraldehyde-3-phosphate dehydrogenase parameters from Teusink <i>et al. </i><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002483#pcbi.1002483-Teusink1" target="_blank">[12]</a>. The concentrations at time zero equal the measured intracellular concentrations. Abbreviations as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002483#pcbi-1002483-g001" target="_blank">Figure 1</a>.</p

Measured fluxes into the side branches and concentrations of allosteric regulators and adenine nucleotides.

<p>The fluxes to (if positive)/from (if negative) trehalose, to glycerol and to succinate are given in mM.min⁻¹ under the growth and starvation conditions studied. These fluxes and metabolite concentrations were subsequently used as fixed parameters in the model described in this study. Positive values indicate fluxes away from glycolysis. Data from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002483#pcbi.1002483-vanEunen3" target="_blank">[23]</a>.</p