Channeling of TCA cycle intermediates in cultured Saccharomyces cerevisiae.
Oxidation of [3-13C]propionate was studied in cultured yeast cells, and the distribution of label in the 2- and 3-positions of alanine was detected by 13C NMR. [3-13C]Propionate forms [2-13C]succinyl-CoA in the mitochondria which then enters the citric acid cycle and forms malate through two symmetrical intermediates, succinate and fumarate. If these symmetrical intermediates randomly diffuse from one enzyme to the next in mitochondria as is normally assumed, then 13C labeling in malate C2 and C3 must be equal. However, any direct transfer of metabolites from site to site between succinate thiokinase, succinate dehydrogenase, and fumarase would result in an uneven distribution of 13C in malate C2 and C3 and any molecules derived from malate. Since pyruvate may be derived from malate via the malic enzyme and subsequently converted into alanine by transamination, any 13C asymmetry in alanine C2 and C3 must directly reflect the 13C distribution in the malate pool. During oxidation of [3-13C]propionate, we detect a significant quantity of labeled alanine, where 13C enrichment in C3 is significantly higher than that in C2. Inhibition of succinate dehydrogenase with malonate or creating conditions that increase the chances of a back-reaction (from malate to fumarate) result in a significant decrease in the asymmetric labeling of alanine. Ubiquinone-deficient yeast cells (having only 10% of the oxidative capacity of wild-type cells) could slowly oxidize propionate, but in this case the 13C labeling was equal in the C2 and C3 of alanine, showing that isotope randomization had occurred.(ABSTRACT TRUNCATED AT 250 WORDS)
Sumegi
B
B
Department of Chemistry, University of Texas, Dallas, Richardson 75083-0688.
Sherry
A D
AD
Malloy
C R
CR
D000214:Acyl Coenzyme A / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D002247:Carbon Isotopes; D002952:Citric Acid Cycle; D009682:Magnetic Resonance Spectroscopy; D008293:Malates / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone); D011422:Propionates / Q000378:metabolism; D011808:Quinone Reductases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D014451:Ubiquinone / Q000494:pharmacology
D000214:Acyl Coenzyme A; D002247:Carbon Isotopes; D008293:Malates; D011422:Propionates; D014451:Ubiquinone; C012046:succinyl-coenzyme A; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; D000409:Alanine