Fig. 1.

Methods of identifying substrate channelling. a Reaction scheme and depiction of transient time (t) analysis based on data from a channelled bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) and a freely diffusing monofunctional TS and DHFR (data from ref. ⁶⁴). b Comparison of residual activity of a channelled or freely diffusing enzyme pair in the presence of a competing enzyme, for example, the malate dehydrogenase and citrate synthase couple in the presence or absence of aspartate aminotransferase, which competes for the metabolic intermediate oxaloacetate (data from ref. ⁸²). c Comparison of residual activity of a channelled or freely diffusing enzyme pair in the presence of an inhibitor of the second enzyme, for example, the inhibition of the TS-DHFR cascade by the inhibition of DHFR by pyruvate (data from ref. ⁶⁴). d Schematic representation of the isotope dilution experiment to assess the channelling of citrate and 2OG. ¹³C-labelled pyruvate was fed to isolated potato mitochondria and the label accumulation in succinate was monitored. The TCA cycle was inhibited by malonate to avoid the complication of multiple turns of the cycle. Non-labelled citrate and 2OG were added into the medium following the fractional enrichment in succinate reaching steady state. Asterisks show the expected fate of labelled carbon following the metabolism of pyruvate under the experimental conditions. e The result of isotope dilution experiments for citrate and 2OG. The time course plots show the fractional ¹³C enrichment in succinate following the addition of unlabelled citrate or 2OG at 0 min. The line is the smoothed conditional mean with the shadow representing a 95% confidence interval. The metabolite is considered not to be channelled when the confidence interval line falls below 0. Panels a–c has been adapted with permission from ref. ¹⁰. Panels d, e has been reproduced with permission from ref. ⁴¹