Evidence for Channeling of Intermediates in the Oxidative Pentose Phosphate Pathway by Soybean and Pea Nodule Extracts, Yeast Extracts, and Purified Yeast Enzymes

Abstract

Evidence is presented that intermediates of the oxidative pentose phosphate pathway (OPPP) are channeled from one pathway enzyme to the next. CO, produced from [l-'4C]glucose in the presence of unlabelled pathway intermediates contained much more radioactivity than predicted by a model in which pathway-produced intermediates are in equilibrium with identical molecules in the bulk phase. This was the case whether glucose 6-phosphate (Glc6P), 6-phosphogluconolactone, or 6-phosphogluconate was added. Assumptions involved in calculating the amount of I4CO, predicted for free mixing of I4C-labelled and unlabelled intermediates are discussed, together with the following results. (a) 14C0, production by pea nodules in the presence of 3 mM 6-phosphogluconate was higher than in its absence. (b) Apparent channeling of intermediates was much higher for purified yeast enzymes than for yeast extract. (c) 6-Phosphogluconate and 6-phosphogluconolactone were channeled between yeast Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase despite the absence of 6-phosphogluconolactonase in the purified yeast enzyme mixture. (d) When purified yeast hexokinase was physically separated from Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase by a dialysis membrane, there was no apparent channeling. (e) Poly(ethy1ene glycol), high salt and detergents had little effect on apparent channeling of OPPP intermediates, which is consistent with a stable complex of enzymes. On the other hand, density gradient centrifugation experiments suggested a more transient interaction between the enzymes. Taken together, the results support channeling of OPPP pathway intermediates.

6-Phosphogluconate Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Form a Supramolecular Complex in Human Neutrophils That Undergoes Retrograde Trafficking during Pregnancy

“…The proximity of these two enzymes suggests that they form a supramolecular complex or metabolon within cells. This suggestion is in agreement with a previous biochemical study in plant and yeast systems using radiolabeled substrates that showed substrate channeling in the HMS (46). Hence, supramolecular complex formation explains FIGURE 11.…”

Changes of Pentose Phosphate Pathway Flux in Vivo in Corynebacterium glutamicum during Leucine-Limited Batch Cultivation as Determined from Intracellular Metabolite Concentration Measurements

“…/mben.2002 hydrolysis of the intermediate 6-phosphoglucono-d-lactone to 6-phosphogluconate (Debnam et al, 1997;Levy, 1979), and in line with our previous publication on this subject (Moritz et al, 2000), we feel it is safe to assume that the glucose-6-phosphate dehydrogenase is in fact the ratecontrolling step of the oxidative part of the pentose phosphate pathway (Greenbaum et al, 1971;Gumaa and Mc Lean, 1969). Therefore only this enzyme is considered in the following discussions on PPP flux.…”

Possible Pitfalls of Flux Calculations Based on 13C-Labeling

“…They found evidence of complexes between g3p dehydrogenase, transketolase, and transaldolase. Debnam et al (1997) concluded from their research that intermediates of the oxidative branch of the PPP are channeled between hexokinase, g6p dehydrogenase, and 6pg dehydrogenase. Further channeling complexes have been suggested between aldolase and glycerol phosphate dehydrogenase, between aspartate aminotransferase and glutamate dehydrogenase (Ova di, 1990), and between the enzymes of the tricarboxylic acid cycle (Haggie et al, 1999).…”