Question

The sulfhydryl group of coenzyme A plays an important role in the biosynthesis of fatty acids from units of acetic acid. The key steps are suggested to be carboxylation with carbon dioxide of acetyl coenzyme $\mathrm{A}$ (which is a thioester abbreviated as O=CSC(=O)O to give malonyl coenzyme $\mathrm{A}$ followed by condensation of the malonyl coenzyme A with acetyl coenzyme $\mathrm{A}$, or a molecule of an acyl coenzyme $\mathrm{A}$, to produce an acyl malonyl coenzyme $\mathrm{A}$ This is followed by decarboxylation and reduction to an acyl coenzyme $A$ with two more carbon atoms than the starting acyl coenzyme $\mathrm{A}$. Each repetition of the process gives a fatty acid (or thioester of a fatty acid) with an additional two carbons. All the steps are of course controlled by specific enzymes. $5^{\prime }-$ (adenosine 3' -phosphate) $-\mathrm{OP}(\mathrm{O})(\mathrm{OH})\mathrm{OP}(\mathrm{O})(\mathrm{OH}){\mathrm{OCH}}_2\mathrm{C}{\left({\mathrm{CH}}_3\right)}_2$ $\mathrm{CH}(\mathrm{OH})-\mathrm{CONH}{\left({\mathrm{CH}}_2\right)}_2\mathrm{CONH}{\left({\mathrm{CH}}_2\right)}_2\mathrm{SH}$ coenzyme $\mathrm{A}(\mathrm{CoA}$ SH ) (a) Write out the complete structure of coenzyme A as corresponds to the above condensed formula; what products would you expect coenzyme $\mathrm{A}$ to give on vigorous hydrolysis? (b) Work out the steps for synthesis of hexanoic acid from acetic acid with the acid of coenzyme $A$, using the abbreviation CoA-SH and indicating the general nature of each step in terms of the kind of reagents that might be used to achieve it in ordinary laboratory operations. What advantages would you expect to accrue from CoA-SH as compared to CoA-OH as the acyl carrier? (c) Suppose the enzymic synthesis of hexanoic acid to be carried on in the presence of radioactive carbon dioxide; would the postulated course of the reaction be precluded if the hexanoic acid produced were completely devoid of radioactivity? Explain in detail.

Short answer

The complete structure of coenzyme A (CoA) is given by the formula: $$5^{\prime }-\text{Adenosine}-3^{\prime }-\text{phosphate}-{\text{OP( O)( OH) OP(O)( OH) OCH}}_2{\text{C(CH}}_3\text{)CH( OH)-}{\text{CONH(CH}}_2{\text{)CONH(CH}}_2\text{)SH}$$ Upon vigorous hydrolysis, CoA breaks down into adenosine, orthophosphate, 2-pyrophosphate, and a sulfhydryl-containing compound. The synthesis of hexanoic acid from acetic acid with coenzyme A involves the following steps: formation of Acetyl-CoA, carboxylation to form Malonyl-CoA, condensation to form acyl-malonyl CoA, decarboxylation and reduction to form a fatty acid with additional carbon atoms. CoA-SH is a better acyl carrier than CoA-OH because it provides more reactive and energy-rich intermediates, facilitating the biosynthesis process. If hexanoic acid synthesis is carried out in the presence of radioactive carbon dioxide and the final product is devoid of radioactivity, it implies that the radioactive carbon is released during the decarboxylation step and does not preclude the postulated course of the reaction.

Step-by-step solution

Write out the structure of coenzyme A

Based on the condensed formula given, the complete structure of coenzyme A (CoA) is as follows: $$5^{\prime }-\text{Adenosine}-3^{\prime }-\text{phosphate}-{\text{OP( O)( OH) OP(O)( OH) OCH}}_2{\text{C(CH}}_3\text{)CH( OH)-}{\text{CONH(CH}}_2{\text{)CONH(CH}}_2\text{)SH}$$

Predict products of CoA on vigorous hydrolysis

Upon vigorous hydrolysis, coenzyme A (CoA) would break down into its constituent components: adenosine, phosphate groups, and the sulfhydryl group. The products formed would be adenosine, orthophosphate, 2-pyrophosphate, and the sulfhydryl containing compound.

Identify steps in the synthesis of hexanoic acid from acetic acid

To synthesize hexanoic acid from acetic acid with the help of coenzyme A, we can follow these general steps: 1. Formation of Acetyl-CoA: Acetic acid reacts with CoA-SH to form Acetyl-CoA (Acetyl thioester of coenzyme A). 2. Carboxylation: Acetyl-CoA reacts with carbon dioxide (CO2) to form Malonyl-CoA, where an extra carbon is added to the acetyl group. 3. Condensation: Malonyl CoA reacts with another Acetyl-CoA (or a molecule of acyl-CoA) to form an acyl-malonyl CoA. 4. Decarboxylation: Acyl-malonyl CoA undergoes decarboxylation, removing the extra carbon added earlier and forming an acyl-CoA with two more carbon atoms than the starting acyl-CoA. 5. Reduction: Acyl-CoA is reduced to form a fatty acid with an additional two carbon atoms. Each repetition of steps 2-5 will convert the starting acetyl-CoA to a longer-chain fatty acid with two additional carbon atoms.

Compare CoA-SH and CoA-OH as acyl carriers

CoA-SH acts as a good acyl carrier due to the presence of the reactive sulfhydryl (-SH) group, which makes the formation of acyl-CoA thioester derivatives more facile. Using CoA-OH would involve ester linkages, which are less reactive and less energy-rich than thioester linkages. Thus, CoA-SH is a better acyl carrier than CoA-OH because it provides more reactive and energy-rich intermediates, facilitating the biosynthesis process.

Analyze radioactivity in hexanoic acid synthesis

If the enzymatic synthesis of hexanoic acid is carried out in the presence of radioactive carbon dioxide, it is expected that the radioactive carbon would be incorporated into malonyl-CoA since this molecule is formed by carboxylation of acetyl-CoA with carbon dioxide. However, if the final hexanoic acid product is completely devoid of radioactivity, it suggests that the radioactive carbon was released during the decarboxylation step. This implies that the radioactive carbon is not a part of the final hexanoic acid product and does not preclude the postulated course of the reaction.

Key concepts

Coenzyme A Structure

Coenzyme A (CoA) is a vital molecule involved in biochemical reactions, especially in fatty acid biosynthesis. It acts as a carrier for acetyl and other acyl groups. CoA is a complex molecule consisting of several functional components: an adenosine 3'-phosphate, a pantothenic acid moiety, a cysteamine group, and a reactive sulfhydryl (thiol) group (-SH). This sulfhydryl group is crucial because it forms thioester bonds with acyl groups.
This structure allows CoA to perform its role effectively in transferring acetyl and acyl groups during metabolic processes. Its ability to form and break these thioester bonds makes it a dynamic participant in several enzymatic reactions.

• 5'-Adenosine: a nucleotide providing the binding site for enzymes.
• Pantothenic acid: a form of vitamin B5 important for coenzyme function.
• Cysteamine group: includes the essential thiol (-SH) part of CoA.

Acetyl-CoA

Acetyl-CoA is a central metabolite in many biochemical pathways, acting as an intersection that connects different biological processes. It is formed by the combination of an acetyl group with coenzyme A, creating a thioester bond. This compound is not just pivotal in fatty acid metabolism but also plays a role in the citric acid cycle and in acetylation of molecules.
In the context of fatty acid biosynthesis, acetyl-CoA provides the two-carbon building blocks needed for the synthesis of fatty acids. Its high-energy thioester bond makes the acetyl group readily transferred to other molecules, facilitating biosynthesis reactions.

• Central to energy production and biosynthesis.
• Contains an acyl group bonded to CoA, crucial for enzymatic bonding activities.
• Links various metabolic pathways within the cell.

Carboxylation Reaction

The carboxylation reaction is a key step in fatty acid biosynthesis, adding a carbon dioxide molecule to an acetyl group to form malonyl-CoA. This reaction is catalyzed by the enzyme acetyl-CoA carboxylase and requires ATP for the energy to form the new carbon-carbon bond.
The addition of CO₂ is essential because it activates the acetyl group, allowing it to participate in the subsequent condensation reaction. Once formed, malonyl-CoA acts as a donor of two carbon units during the chain elongation of fatty acids.

• Involves the addition of CO₂ to acetyl-CoA.
• Results in the formation of malonyl-CoA, a key intermediate.
• This step is ATP-dependent and requires enzymatic activity.

Thioester Linkage

Thioester linkage is a critical chemical bond that forms between the sulfhydryl group of CoA and an acyl group. In acetyl-CoA and other similar compounds, this linkage is essential for high-energy transfer reactions.
The thioester bond is more reactive than regular ester bonds, which makes it ideal for energy-requiring cellular processes. During fatty acid synthesis, the energy from breaking the thioester bond is utilized to drive the formation of longer-chain fatty acids.

• Characterized by a bond between a sulfur atom and a carbonyl group.
• High energy and reactivity make it suitable for biosynthetic pathways.
• Enables the transfer of acyl groups in metabolic processes.

Decarboxylation Process

The decarboxylation process is a crucial step during the elongation of fatty acids. Once malonyl-CoA condenses with an acetyl group, the resulting acyl-malonyl CoA undergoes decarboxylation. This means it loses a carbon dioxide molecule, leaving behind an acyl group elongated by two carbon atoms.
This step effectively removes the same carbon dioxide that was initially added during the carboxylation step. It helps drive the reaction forward and is crucial for the synthesis of longer fatty acid chains. By removing the CO₂, the stability and directionality of the biosynthesis process are maintained.

• Involves the release of CO₂ from a compound.
• Critical for extending carbon chains during fatty acid synthesis.
• Ensures the continuation and completion of the fatty acid biosynthesis process.