CHEM-642 Biochemistry
Practice Problem

Contemporary Approaches for Determining Metabolic Pathways:
13C NMR and Riboflavin Biosynthesis

Textbooks and metabolic maps often give the mistaken impression that all of the pathways of intermediary metabolism are known. Actually, there are many well-known biomolecules for which the biosynthetic pathways are poorly understood or never studied. For example, the origin of the eight-carbon atoms comprising the xylene-ring of riboflavin only recently was deduced (1). The classical approach using radiolabeled precursors required complicated chemical degradation procedures to locate labeled atoms. This tedious and difficult procedure, once worthy of Nobel prizes (eg. Bloch 1964), has given way to a much preferred tracer strategy.

Carbon 13 is a non-radioactive isotope of carbon. In contrast to carbon 12, this isotope has a nuclear spin that can be detected by nuclear magnetic resonance (NMR). Because the chemical environment of an atomic nucleus in influences it chemical shift in NMR, most is not all carbon atoms in an organic molecule can be identified nondestructively. Furthermore, the natural abundance of 13C is only 1.1%, thus 13C-enrichment from various precursors can be detected easily in an isolated product.

All of the resonances in the 13C-NMR spectrum of riboflavin have been assigned and are indicated in the table below. Also shown are the relative 13C-enrichment for each carbon atom of riboflavin after feeding the indicated 13C-precursors. As with any experiment of this type, there will be dilution of the isotope by endogenous precursors and product. These experiments were done with the mold Ashbya gossypii.
 

Relative 13C-enrichments of riboflavin tetraacetates obtained from various 13C-labeled precursors
 

Carbon Atom 
in Riboflavin

Chemical Shift
ppm
Relative Enrichments
[1-13C]
Acetate
[2-13C]
Acetate
[1-13C]
Ribose
[1-13C]
Glucose
[6-13C]
Glucose
[2-13C]
Glycerol
2 154.9 1.8 2.7 0.9 1.1 1.6 5.4
4 159.5 4.9 1.9 0.8 1.2 1.2 1.4
4a 136.0 0.9 0.6 0.9 1.3 1.1 1.9
5a 136.6 6.3 5.2 3.3 2.4 2.2 15.9
6 132.8 3.3 5.8 10.3 5.5 3.2 12.9
7 137.1 8.3 3.0 0.8 1.4 1.6 3.0
7a 19.5 1.2 7.0 0.9 3.3 9.0 2.4
8 148.1 5.5 3.6 3.7 1.8 1.9 12.1
8a 21.5 3.3 6.0 9.1 5.0 2.7 12.3
9 115.6 1.2 6.4 1.1 3.4 9.0 2.1
9a 131.2 8.5 2.1 0.6 1.2 1.6 3.0
10a 150.6 1.3 0.8 0.6 0.7 0.9 1.1
1' 45.0 3.2 4.6 19.4 4.5 2.3 8.5
2' 69.4 4.4 4.0 2.9 1.8 1.6 7.4
3' 70.4 6.9 2.7 0.6 1.5 1.7 2.1
4' 69.0 1.0 4.9 1.3 1.9 1.9 12.9
5' 61.9 1.1 4.7 1.1 3.2 6.8 1.6
CH3CO 20.4-21.1 1.0 1.0 1.0 1.0 1.0 1.0
CH3CO 169.8-170.7 1.0 0.8 0.9 0.9 0.8 0.9
The final two entries are from the four acetyl groups on the tetraacetylated riboflavin.  The values are the average of the four signals and serve as a internal, natural abundance standard.

 

 
 
 
 
 

        GTP

Questions:

1.  GTP is a precursor of riboflavin. Draw the structure of GTP in the space above next to riboflavin so that the structural relationships can be readily appreciated.  Try to deduce which atoms of riboflavin are derived from GTP and which are added.

2.  Examine the 13C-labeling pattern for riboflavin from the various labeled precursors. Is the labeling pattern of those carbons derived from GTP consistent with the known biosynthetic pathway for GTP? Justify your answer.

3.  Using circles, squares, triangles, etc. or different colors drawn on the structure above, identify carbon atoms in riboflavin that have a common pattern (origin).

4.  Based on the distribution of label in your answer to Question 3, what can you deduce about the biosynthesis of the xylene ring moiety of riboflavin?

5.  Considering the coupling of nuclear spins detectable by NMR, what biosynthetically important information could be obtained by comparing the 13C-NMR spectra of riboflavin synthesized from [4-13C] or [5-13C] Ribose versus that synthesized from [4,5-13C] Ribose? Here [4,5-13C] Ribose is doubly labeled, not a mixture of [4-13C] and [5-13C] Ribose.)



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Last updated: 3 February 2001 by Hal White
Copyright 2000, Department of Chemistry and Biochemistry, University of Delaware, Newark, DE  19716