Question

Using your knowledge of fatty acid biosynthesis, provide an explanation for the two experimental observations. a. A biochemist adds uniformly labeled $\left[{}^{14}\mathrm{C}\right]$ acetyl-CoA to a soluble liver fraction, which yields palmitate uniformly labeled with ${}^{14}\mathrm{C}$. b. In a second experiment, the biochemist adds a trace of uniformly labeled ${}^{[14}\mathrm{C}]$ acetyl-CoA in the presence of an excess of unlabeled malonyl-CoA to a soluble liver fraction, which yields palmitate labeled with ${}^{14}\mathrm{C}$ only in C-15 and C-16.

Short answer

Observation (a): Uniformly labeled due to all precursors being labeled acetyl-CoA. Observation (b): Labeled at C-15, C-16 due to trace labeled acetyl-CoA in the presence of excess unlabeled malonyl-CoA.

Step-by-step solution

Understand Fatty Acid Biosynthesis

Fatty acid biosynthesis involves the repetitive addition of two-carbon units, derived from acetyl-CoA, to a growing fatty acid chain. The process occurs in a series of steps catalyzed by fatty acid synthase. Malonyl-CoA is the primary donor of two-carbon units in the chain elongation process, contributing carbons through a decarboxylation reaction.

Analyze Observation A

In observation (a), the uniform labeling of palmitate with ${}^{14}\mathrm{C}$ from acetyl-CoA suggests that all carbon atoms in the resultant palmitate originate from labeled acetyl-CoA. Normally, acetyl-CoA contributes as the primer at the initial (C-1 and C-2) positions and then as a two-carbon provider with malonyl-CoA unit decarboxylation. Here, since all precursors are labeled, every carbon incorporated into palmitate will have ${}^{14}\mathrm{C}$.

Analyze Observation B

In observation (b), the trace of ${}^{14}\mathrm{C}$ labeled acetyl-CoA in the presence of an excess of unlabeled malonyl-CoA results in palmitate labeled only at C-15 and C-16. This is because the labeled acetyl-CoA is used at the starting point of synthesis but diluted with numerous cycles of unlabeled malonyl-CoA additions. Thus, only the final condensation reaction next to the terminal gives rise to ${}^{14}\mathrm{C}$ in the elongation of the fatty acid chain.

Conclusion

The observations demonstrate how the source of the ${}^{14}\mathrm{C}$ label influences its incorporation into the fatty acid. Experiment (a) results in uniform labeling due to the exclusive use of labeled acetyl-CoA, while experiment (b) shows labeling predominantly in the last two positions due to the relatively limited involvement of labeled acetyl-CoA in the presence of excess unlabeled precursors.

Key concepts

acetyl-CoA

Acetyl-CoA is a central molecule in the metabolism of living cells. It is a versatile building block that contributes to synthesizing many important compounds. In fatty acid biosynthesis, acetyl-CoA plays a critical role by serving as the starting material for the creation of new fatty acids. It transfers a two-carbon unit to begin the fatty acid synthesis process.

Key functions of acetyl-CoA in fatty acid biosynthesis include:

• Initiator: Acetyl-CoA provides the initial two-carbon unit required to start the creation of a fatty acid chain. This is the primer that sets the synthesis in motion.
• Carbon Provider: While acetyl-CoA begins the process, it also continues to offer two-carbon groups that help elongate the growing fatty acid chain in collaboration with malonyl-CoA.
• Participator in the Citric Acid Cycle: Beyond fatty acid biosynthesis, it is essential in energy production in the citric acid cycle.

Understanding acetyl-CoA's role provides insight into how fatty acids are efficiently synthesized, showing why it is vital in both providing raw materials and helping energy production.

malonyl-CoA

Malonyl-CoA is a derivative of acetyl-CoA and plays a distinct and significant role in fatty acid biosynthesis. It serves as the primary extender unit for the growing fatty acid chain.

Let's explore the role of malonyl-CoA in this process:

• Extension of Carbon Chain: Malonyl-CoA contributes to the elongation of the fatty acid chain by providing two-carbon units. It adds to the chain through a process known as decarboxylation, where it loses a carbon dioxide molecule, enriching the chain link by link.
• Regulation: Malonyl-CoA also acts as a regulatory molecule, controlling the synthesis and degradation of fatty acids. Its concentration influences the switch between building new fatty acids or breaking them down for energy.
• Facilitator: The reaction involving malonyl-CoA is catalyzed by fatty acid synthase, and without malonyl-CoA, the enzyme cannot extend the chain, highlighting its essential role in this enzymatic process.

By understanding malonyl-CoA's function, you grasp why it is indispensable in the biosynthesis of fatty acids, acting as an extender and regulator in the process.

palmitate labeling

Palmitate labeling in biochemical experiments helps scientists understand the carbon source and pathway in fatty acid biosynthesis. It involves tracking the incorporation of carbon atoms into palmitate, a common long-chain fatty acid.

When analyzing palmitate labeling, consider these points:

• Source Identification: By using labeled carbon atoms, such as ${}^{14}\mathrm{C}$, scientists can determine which carbon sources (acetyl-CoA or malonyl-CoA) are contributing to the construction of the palmitate molecule.
• Clarifying Pathways: Observation of labeled positions within palmitate provides insight into which enzymes and pathways are active during its synthesis. For example, uniform labeling across all carbon atoms indicates that the source acetyl-CoA contributes evenly throughout the process.
• Experiment Interpretation: By comparing different labeled and unlabeled experiments, one can deduce how acetyl-CoA and malonyl-CoA interact, as demonstrated by labeling only in specific carbon positions like C-15 and C-16.

Palmitate labeling reveals the detailed contributions of these molecules, shedding light on the dynamics of fatty acid biosynthesis.

fatty acid synthase

Fatty acid synthase is an essential enzyme complex responsible for building fatty acids from acetyl-CoA and malonyl-CoA. It operates through a series of coordinated steps and biochemical reactions.

Consider these important roles of fatty acid synthase in fatty acid biosynthesis:

• Enzymatic Activity: It houses various enzyme activities within a single polypeptide chain that work in tandem to catalyze the reactions needed for fatty acid chain elongation.
• Efficient Process: By organizing multiple enzyme activities, fatty acid synthase ensures that the fatty acid synthesis proceeds with precision and efficiency, reducing the loss of intermediate molecules.
• Coordination: It coordinates the condensation, reduction, dehydration, and reduction reactions that incrementally extend the fatty acid chain by repeating cycles until a fatty acid like palmitate is fully formed.

Understanding fatty acid synthase allows appreciation of how these complex, yet efficient processes orchestrate the synthesis of essential fatty acids, central to cellular functions.