The chemistry of most metabolic reactions was deciphered by synthesizing metabolites containing atoms that are different isotopes from those occurring naturally. The products of reactions starting with isotopically labeled metabolites can be analyzed to determine precisely which atoms in the products are derived from which atoms in the starting material. The methods of detection exploit, for example, the fact that different isotopes have different masses that can be distinguished using biophysical techniques such as mass spectrometry. Moreover, some isotopes are radioactive and can therefore be readily recognized with electronic counters or photographic film that becomes exposed by radiation.

A. Assume that pyruvate containing radioactive 14C in its carboxyl group is added to a cell extract that can support oxidative phosphorylation. Which of the molecules produced should contain the vast majority of the 14C that was added?

B. Assume that oxaloacetate containing radioactive 14C in its keto group (refer to Panel 13–2, pp. 442 443) is added to the extract. Where should the 14C atom be located after precisely one turn of the citric acid cycle.

Panel 13–2

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pyruvate CH3 COO NAD+ COO™ H-C-OH CO₂ STEP 7 NAD+ NADH NADH H* STEP 8 CH₂ malate 4C coo H₂O HS-COA coenzyme A fumarate 4C COO™ CH 11 CH coo COO ¢=0 CH₂ coo oxaloacetate 4C next cycle STEP 6 FADH₂ FAD acetyl CoA 2C COO C=O CH₂ Coo™ oxaloacetate 4C CH3-C-S-COA succinate 4C COO™ CH₂ CH₂ Coo CITRIC ACID CYCLE HS-CoA H₂O STEP 1 STEP 5 GTP HS-COA GDP COO™ CH₂ HO-Ć-COO™ CH₂ Coo- citrate 6C succinyl CoA 4C COO™ H₂O Overview of the complete citric acid cycle. The two carbons from acetyl CoA that enter this turn of the cycle (shadowed in red) will be converted to CO₂ in subsequent turns of the cycle: the two carbons in the starting oxaloacetate (shadowed in blue) will be converted to CO₂ in this cycle. CH₂ CH₂ c=o I S-CoA STEP 2 isocitrate 6C HO-CH a-ketoglutarate 5C I STEP 4 NAD COO™ I CH₂ HC-COO™ STEP 3 COO coo™ NADH + H CH₂ I CH₂ c=0 Coo- HS-COA NAD NADH + H+