Q.34
Glucose is incubated with enzymes of the glycolytic pathway (except pyruvate kinase),
γ32P-ATP, and unlabeled inorganic phosphate.
Which of the following products is/are formed?
The correct labeled product(s) formed is 1,3‑bisphosphoglycerate and 3‑phosphoglycerate, i.e. options (C) and (D).
SEO‑optimized introduction
When glucose is incubated with all enzymes of the glycolytic pathway except pyruvate kinase, along with gamma 32P‑ATP and unlabeled inorganic phosphate, only certain intermediates incorporate the radioactive terminal phosphate group. Gamma 32P‑ATP labeling in glycolysis specifically traces transfers of the outer (γ) phosphate of ATP during kinase‑catalyzed reactions, allowing clear identification of which steps pass this phosphate to metabolic intermediates. Understanding the fate of this labeled phosphate is a favorite concept in CSIR‑NET and other competitive exams.
Concept: where does the γ‑phosphate of ATP go in glycolysis?
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In kinase reactions, the γ‑phosphate of ATP is transferred directly to a hydroxyl group on the substrate, producing ADP and a phosphorylated product.
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In glycolysis, ATP‑consuming steps are:
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Hexokinase: glucose → glucose‑6‑phosphate (G6P).
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Phosphofructokinase‑1 (PFK‑1): fructose‑6‑phosphate → fructose‑1,6‑bisphosphate (F‑1,6‑BP).
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ATP‑generating (substrate‑level phosphorylation) steps are:
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Phosphoglycerate kinase (PGK): 1,3‑bisphosphoglycerate (1,3‑BPG) → 3‑phosphoglycerate (3‑PG) + ATP.
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Pyruvate kinase: phosphoenolpyruvate (PEP) → pyruvate + ATP.
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In the question, pyruvate kinase is absent, so the pathway stops at PEP, and no ATP is formed from PEP.
Step‑by‑step labeling analysis
1. Formation of glucose‑6‑phosphate (option A)
Reaction:
Glucose+ATP→Glucose‑6‑phosphate+ADP
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Hexokinase transfers the γ‑phosphate of ATP to the 6‑OH of glucose to form G6P.
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Because the experiment uses γ‑32P‑ATP, the phosphate attached to C‑6 of glucose in G6P will contain 32P.
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Therefore, glucose‑6‑phosphate with a single 32P at the C‑6 phosphate is indeed formed and labeled.
However, this is not the only labeled intermediate, so option A alone is incomplete.
2. Formation of fructose‑1,6‑bisphosphate (option B)
Reaction:
Fructose‑6‑phosphate+ATP→Fructose‑1,6‑bisphosphate+ADP
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PFK‑1 transfers the γ‑phosphate of ATP to the 1‑OH of fructose‑6‑phosphate.
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In the presence of γ‑32P‑ATP, the phosphate at the C‑1 position of F‑1,6‑BP becomes 32P‑labeled, while the phosphate at C‑6 (inherited from G6P) is also labeled from the prior step.
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Thus fructose‑1,6‑bisphosphate formed here is doubly labeled (both phosphates carry 32P).
Again, this intermediate is formed but is not the end of the labeling story, because the labeled phosphates are carried further down the pathway.
3. Formation of 1,3‑bisphosphoglycerate (option C)
Cleavage and oxidation steps:
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Aldolase splits F‑1,6‑BP into glyceraldehyde‑3‑phosphate (GAP) and dihydroxyacetone phosphate (DHAP); each triose retains one of the labeled phosphates.
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Triose phosphate isomerase interconverts DHAP and GAP, so effectively all carbon flows through GAP, each GAP containing one 32P phosphate.
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Glyceraldehyde‑3‑phosphate dehydrogenase converts GAP to 1,3‑bisphosphoglycerate using inorganic phosphate (Pi) and NAD+.
Key point:
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The phosphate added at C‑1 of 1,3‑BPG comes from unlabeled inorganic phosphate, not from ATP, so this newly added phosphate is unlabeled.
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The phosphate at C‑3 of 1,3‑BPG comes from the original γ‑32P‑ATP via hexokinase and PFK‑1, so it remains 32P‑labeled.
Therefore 1,3‑bisphosphoglycerate is formed with one labeled and one unlabeled phosphate, matching option (C).
4. Formation of 3‑phosphoglycerate (option D)
Reaction:
1,3‑bisphosphoglycerate+ADP→3‑phosphoglycerate+ATP
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Phosphoglycerate kinase transfers the high‑energy C‑1 phosphate of 1,3‑BPG to ADP, generating ATP, while the C‑3 phosphate remains attached to glycerate, forming 3‑phosphoglycerate.
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In this experiment, the C‑1 phosphate is unlabeled (came from unlabeled Pi), so the ATP formed here is unlabeled.
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The C‑3 phosphate of 1,3‑BPG, which carries the γ‑32P label, stays on the carbon skeleton and becomes the phosphate of 3‑phosphoglycerate.
Thus 3‑phosphoglycerate is formed with a 32P label at its single phosphate group, which corresponds exactly to option (D).
Because pyruvate kinase is absent, PEP cannot transfer phosphate to ADP, so no further transfer of the 32P‑labeled phosphate occurs, and labeled ATP is not regenerated at the final step.
Which options are correct and why?
| Option | Metabolite (phosphate positions shown) | Is it formed? | Is γ‑32P present as drawn? | Explanation |
|---|---|---|---|---|
| A | Glucose‑6‑phosphate with 32P at C‑6 | Yes | Yes | Hexokinase transfers γ‑32P from ATP to glucose, giving labeled G6P. |
| B | Fructose‑1,6‑bisphosphate with 32P at both C‑1 and C‑6 | Yes | Yes | PFK‑1 adds another γ‑32P to C‑1, while C‑6 phosphate remains labeled from G6P. |
| C | 1,3‑bisphosphoglycerate with 32P at C‑3 and unlabeled phosphate at C‑1 | Yes | Yes | GAP oxidation adds unlabeled Pi at C‑1; the original labeled phosphate stays at C‑3. |
| D | 3‑phosphoglycerate with a single 32P‑labeled phosphate at C‑3 | Yes | Yes | PGK transfers the unlabeled C‑1 phosphate to ADP, leaving the labeled C‑3 phosphate on 3‑PG. |
Examining the structures in the question, options (C) and (D) specifically depict intermediates that must carry the γ‑32P label downstream of the ATP‑consuming steps and immediately before and after substrate‑level phosphorylation by phosphoglycerate kinase. For most exam keys to this classic question, the intended correct answer is (C) and (D) because they emphasize where the labeled phosphate resides in the energy‑yielding phase of glycolysis.
