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36: Fatty Acid Pathways and Regulation (Worksheet)

  • Page ID
    81927
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    Name: ______________________________

    Section: _____________________________

    Student ID#:__________________________

    Work in groups on these problems. You should try to answer the questions without referring to your textbook. If you get stuck, try asking another group for help.

    Fatty Acid Biosynthesis The anabolic pathway for fatty acids involves the sequential addition of two carbon units from Acetyl CoA. Acetyl CoA is primarily derived from glycolysis. Thus, dietary carbohydrates can be converted into fats for storage. In both bacteria and eukaryotic cells, the overall reaction converts 8 AcetylCoA (2C) into 1 palmitate, a 16 C fatty acid as shown in the reaction below.

    Fatty acids are made by a repetitive four-step sequence of enzyme-catalyzed.

    1.png

    Acyl Transfers

    The starting material for fatty acid synthesis is the thioester, acetyl CoA which is converted to acetyl ACP (acetyl carrier protein) and then into acetyl synthase (more reactive acetyl group).

    • Draw a mechanism for this trans(thio)esterification.

    2.png

    Carboxylation and Acyl Transfer

    In this step, Acetyl CoA is carboxylated to form the malonyl CoA, catalyzed by the enzyme Acetyl CoA Carboxylase (not part of fatty acid synthase). The malonyl group is then transferred to the empty ACP to form malonyl-ACP. ATP-dependent carboxylation of the biotin, carried out at one active site (1), is followed by transfer of the carboxyl group to acetyl-CoA at a second active site (2).

    • Draw arrows for the proposed mechanism for the carboxylation of acetyl CoA shown below in active site 2.

    3.png

    Following the formation of malonyl CoA, the malonyl is transferred to a carrier protein (in a transesterification step much like acetyl CoA).

    Condensation

    Now both acetyl and malonyl groups are on the carrier proteins and are poised for condensation.

    • Propose a mechanism for this reaction.

    4.png

    Reduction

    The ketone group in acetoacetyl ACP is next reduced to the alcohol by b-keto thioesterase and NADPH.

    • Draw a mechanism for this reaction.

    5.png

    Dehydration

    (This is like the dehydration step of an aldol condensation)

    • Draw a mechanism for this reaction.

    6.png

    Reduction (1,4 addition of the hydride)

    • Draw a mechanism for this reaction.

    7.png

    Summary

    1. Two cycles of Fatty Acid Synthesis:

    8.png

    The net effect of these each turn on the fatty acid spiral is to add another 2 two-carbon groups on to the saturated acyl group. 2. The roadmap shown above is 2 cycles of fatty acid biosynthesis. Draw the product after a 3rd cycle.

    Practice Elongation

    The butyryl ACP is then transferred to the cysteine-SH at the acyl binding site of the ketosynthase enzyme where it undergoes condensation with another malonyl ACP to yield a six-carbon unit. Each repetition of this cycle adds two more carbons until palmitoyl ACP (16C) is formed.

    • Draw another iteration of this pathway to convert the butyryl ACP into the six carbon acyl group.

    Megasynthases: Enzyme Assembly Lines

    Fatty acid synthesis requires many different enzymatic reactions. In mammals, the entire pathway of palmitate synthesis from malonyl-CoA is catalyzed by a single, homodimeric, multifunctional protein, the fatty acid synthase. Current understanding of fatty acid biosynthesis and the acyl carrier protein
 David I. Chan and Hans J. Vogel, 
 Biochem. J. (2010) 430 (1-19)

    13.png

    • Each of the two letter codes stands for one of the enzymes in the pathway. Correlate each code with an enzyme name:

    • MT
    • KS
    • AT
    • ER
    • DH
    • KR

    • What is the advantage of a megasynthase for an organism (Hint: Think of \(\Delta G=\Delta H-T \Delta S\))?

    Triglyceride formation

    The Fatty acids are then converted to triglycerides for easier transport out of the cell. Triglycerides are formed by the combination of glycerol with three fatty acids.

    11.png


    • Circle the three fatty acids.

    Propose a mechanism for the formation of the triglyceride from glycerol (shown below) and palmitoyl CoA (Hint: What is the functional group of palmitoyl-CoA?).

    12.png

    • Compare the polarity of a fatty acid to the triglycerides.

    Many cell types and organs have the ability to synthesize triacylglycerols (TAG), but in animals the liver, intestines and adipose tissue are most active. TAGs are stored as cytoplasmic 'lipid droplets' enclosed by a monolayer of phospholipids and hydrophobic proteins. • Draw a picture of a lipid droplet in a monolayer of phospholipids.

    • Fatty acids are stored when food supplies are ( low OR in excess). • The lipid serves as a store of ___________, which can be released on demand. • Suggest a reason for enclosing the lipids inside a droplet.

    Fatty Acid Catabolism - Now do it in reverse!

    Energy-dense TAG (triacylglycerol) can be mobilized and used for energy during times of low carbohydrate availability (fasting or famine) or during heightened metabolic demand (exercise or cold-stress). The first step of this process is convert the TAGs back to individual fatty acids.

    TAG Lipase

    Triacylglycerols are a major energy dense storage which can be released on demand.

    • Fatty acids are stored when food supplies are (low OR in excess).
    • Fatty acids are released when food supplies are (low OR in excess).

    TAG lipase hydrolyzes the fatty acids from the triacylglycerides.

    • Propose a mechanism for the hydrolysis of a triacylglyceride into fatty acids and glycerol.

    14.png

    B-oxidation of Fatty Acids (Fatty Acid Spiral)

    Beta-oxidation splits long chains of fatty acids into acetyl-CoA molecules in mitochondria and/or in peroxisomes for energy use. This cycle repeats removing two carbon units until the fatty acid has been completely reduced to acetyl-CoA.

    15.png

    Mechanistic View of b-oxidation:

    The first step of this b-oxidation cycle is the formation of an a,b-unsaturated thioester.

    • You will discuss the mechanism in the next course, but for now you can draw arrows to show electron flow in this reaction:

    16.png

    Two electrons from \(FADH_2\) are fed into the mitochondrial electron transport chain that eventually produces ATP. The second step of this b-oxidation cycle is the addition of the elements of \(H_2O\) across the new double bond. The first step in this proposed mechanism is conjugate addition of water.

    • Draw arrows to show electron flow and predict a structure for the intermediate formed:

    17.png

    We have actually seen the third step in this b-oxidation cycle before. Previously we looked at NADH as a hydride reducing agent.

    • Knowing this, propose a mechanism for the following reaction:

    18.png


    • Propose a mechanism for the last step of this b-oxidation cycle (retro-Claisen):

    19.png

    • Why does the CoA-SH attack the ketone and not the thioester?

    Control of Fatty Acid

    Metabolism Anabolism vs. Catabolism

    • Review: Match the terms with the meaning. Anabolic breaking molecules down Catabolic biosynthesis of primary metabolites
    • Match the pathways. Anabolic b-oxidation Catabolic fatty acid synthesis
    • Match the pathways with the use. b-oxidation Fats à Acyl CoA for energy use fatty acid synthesis Acyl CoA à Fats for storage

    Some characteristic features about these competing pathways:

    Difference b-oxidation biosynthesis
    Location mitochondria cytosol
    Acyl Group Carrier CoA ACP
    Redox Enzyme FADH NADPH

    Regulation of Fatty Acid Synthesis

    Fatty acid synthesis occurs only when the organisms’ energy needs have been met. We will look at the regulatory mechanisms that control fatty acid synthesis under conditions of dietary excess.

    • From a biological sense, would you expect fatty acid synthesis to occur when:
      • Available carbohydrates (excess food) are (high or low)?
      • Energy expenditure is (high or low)?
      • Fatty acids levels are (high or low)?
    • Look back at the overview of the full fatty acid pathway. Which enzyme would be the best one to regulate?

    20.png

    Energy Storage vs Energy Usage Acetyl

    CoA can enter into Fatty Acid Synthase to store fat or it can enter the TCA cycle to generate immediate energy for the organism.

    21.png

    • From an efficiency standpoint, why would the fatty acid synthesis be regulated by switching “on” or “off” the enzyme that reacts HCO3- with acetyl CoA to form malonyl CoA

    Control #1: Compartmentalization of pathways

    We have seen several forms of regulation already. Physical segregation of metabolic processes into distinct subcellular locations like the cytosol or specialized organelles (nucleus, endoplasmic reticulum, Golgi apparatus, lysosomes, mitochondria, etc.) is another form of regulation. For instance, glycolysis and fatty acid synthesis take place in the cytosol and the tricarboxylic acid (TCA) cycle and fatty acid oxidation take place in the mitochondria.

    • Why wouldn’t we want to have fatty acid synthesis and fatty acid oxidation occur in the same compartment?

    Control #2: Covalent Modification – Phosphorylation of ACC

    One mechanism to control the activity of Acetyl CoA carboxylase (ACC) is phosphorylation/dephosphorylation of serine side chains (-CH2OH) in the enzyme. AMP-dependent protein kinase (AMPK) catalyzes the covalent transfer of phosphate from ATP to the serine OH groups of the carboxylase.

    • Fill in the boxes on the diagram below.
      • When ACC is active, the cell is producing ATP or storing energy as fats?
      • When ACC is inactive, the cell is producing ATP or storing energy as fats?

    22.png

    Keep in mind, adenosine mono-, di- and triphosphate exist in an interconvertible “pool” in cells:

    \[AMP \rightleftharpoons ADP \rightleftharpoons ATP \]

    When the cell is under stress or exercise, what happens to:

    • ATP levels?
    • AMP levels?

    Explain in your own words why high levels of AMP in the cell would control the AMPK to turn off the Acetyl CoA Carboxylase. What is the end result of ‘turning the switch’?

    Control #3: Allosteric Control – Citrate and Palmitoyl CoA Levels

    Acetyl CoA carboxylase is also regulated by noncovalent binding of citrate, the condensation product of oxaloacetate and acetyl CoA in the TCA cycle.

    • When the energy needs of the cell have been satisfied (but the switch hasn’t turned yet):
      • Citrate levels will start to (increase or decrease)?
      • Acetyl CoA will start to (increase or decrease)?
      • ATP levels will start to (increase or decrease)?

    High levels of mitochondrial citrate lead to the movement of citrate into the cytoplasm.

    • Krebs cycle occurs in the Cytosol or Mitochondria ?
    • Fatty Acid Synthesis occurs in the Cytosol or Mitochondria?

    In the cytoplasm, citrate binds to a site different from the active site and changes the Acetyl CoA carboxylase from a dimer (two interacting carboxylases) to a long filament.

    23.png

    • As citrate builds up, ‘the switch’ is turned which direction? Why?

    The end product of fatty acid synthase (palmitoyl CoA, 16C) also binds to acetyl CoA carboxylase.

    • As palmitoyl CoA builds up, ‘the switch’ is turned which direction? Why?

    Control #4: Hormone Levels

    Acetyl CoA carboxylase can also be regulated by hormones. Hormones usually act by binding to hormone receptors (transmembrane proteins) on the surface of cells, leading to conformational changes in the protein that activate other proteins inside the cell. Insulin is secreted from pancreatic cells in the presence of sugar in the blood after ingestion of food. It signals, in effect, the well-fed state. The hormones, glucagon and epinephrine, signal that energy is in short supply. These different hormones affect acetyl CoA carboxylase.

    • On the diagram below, fill in the boxes with the hormone that activates the enzymes that, in turn, control acetyl Co carboxylase. Explain your choice.


    24.png

    In both type I and II diabetes, the transport of glucose into muscles, liver, and fat tissue is significantly reduced. Despite the abundant amount of glucose in the blood, the cells are metabolically starved.

    • Since these cells are “starved” what can they turn to for an energy source?

    Control #5: Membrane transport

    Not all molecules can passively diffuse across membranes. Often it is the transport of substrates or products into or out of these compartments that is regulated. This is much like when you go to a movie, you need to go through one specified entrance and present a ticket in order to gain entrance. Glucose transport example GLUT4 is the insulin regulated glucose transporter found in adipose tissues and striated muscle (skeletal and cardiac) that is responsible for insulin-regulated glucose translocation into the cell.

    Fletcher, Welsh, Oatey and Tavare, Biochem. J. (2000) 352 (267–276).


    23 - Copy - Copy.png


    Figure 1. Insulin binds to its receptor (1) which in turn starts many protein activation cascades (2). These include: translocation of Glut-4 transporter to the plasma membrane and influx of glucose (3), glycogen synthesis (4), glycolysis (5) and fatty acid synthesis (6).


    • In the example above, what is the “ticket” that must be present to gain entrance into the cell?


    The process is not quite as straight forward as depicted in the figure above. Under conditions of low insulin, GLUT4 is sequestered in intracellular vesicles in muscle and fat cells. Insulin induces a rapid increase in the uptake of glucose by inducing the translocation of GLUT4 from these vesicles to the plasma membrane. As the vesicles fuse with the plasma membrane, GLUT4 transporters are inserted and become available for transporting glucose, and glucose absorption increases. • Draw a cartoon of this translocation process.
    Summary • On the “cartoon” diagram of the cellular regulation of fatty acid synthesis: o Circle the phosphorylated form of ACC. o Does protein kinase activate or deactivate the ACC? o Does glucagon activate or deactivate ACC? o Put a box around the phosphorylated form of TAG lipase. o Does protein kinase activate or deactivate TAG lipase? o Insulin would trigger (release OR storage ) of TAGs. o Glucagon would trigger (release OR storage ) of TAGs.



    ATP
    cAMP
    Adenylyl cyclase
    G-protein
    AMP
    insulinglucagon
    Protein kinase (inactive)
    Protein kinase (active)
    Phosphodiesterase
    Acetyl-CoA carboxylase (active)
    Triacylglycerol lipase (inactive)
    Acetyl-CoA carboxylase (inactive)
    Triacylglycerol lipase (active)
    P
    ATP ADP
    H2OHPO 4-2
    P
    ATPADP
    H2O HPO4-2 Phosphatases
    Protein kinase
    Protein kinase
    Phosphatases
    Triglycerides
    glygerol fatty acids
    Acetyl-CoA
    Malonyl-CoA
    Regulation of Fatty Acid vs. Beta Oxidation: • What is the result of fatty acid synthesis: Fat storage or energy production • What is the goal in beta oxidation? Fat storage or energy production



    • Once the organism commits to pathway A à E by synthesizing B, would compound B be likely to inhibit or activate enzyme Z?

    Assume that fatty acid synthesis is the pathway A à E • What is the product of the first reaction in fatty acid synthesis (B)? • What is the pathway E àA? • What is the first substrate (E) in beta oxidation?

    The beta oxidation of fatty acids involves three stages: Z. Activation of fatty acids in the cytosol Y. Transport of activated fatty acids into mitochondria X. Beta oxidation proper in the mitochondrial matrix

    In the first step of b-oxidation (Z), a transport protein on mitochondrial membranes binds to palmitoyl CoA and moves it into the mitochondria for metabolism. Malonyl CoA is an allosteric regulator of this step. • Is malonyl CoA an activator or inhibitor of this movement? Why?




    A B C D E 1 2 3 4 W X Z Y
    Additional Problems 1. Why do most fatty acids contain an even number of carbons?

    2. The complete oxidation of glucose to CO2 releases -2850 kJ/mol while the oxidation of palmitate releases -9781 kJ/mol. a. Compare the energy per carbon released in the oxidation of both molecules. b. Compare the number of ATP molecules released per carbon in the oxidation of each of these molecules. c. What is the efficiency of oxidizing carbohydrates vs. fats?

    3. Muscles are made up of cells called fast twitch and slow twitch. Slow twitch muscles are suited for endurance and are slow to fatigue because they have additional mitochondria and enzymes for oxidative metabolism to generate ATP. Fast twitch muscles have a greater reliance on glycolytic enzymes. These fibers are efficient for short bursts of speed and power and use both oxidative metabolism and anaerobic metabolism depending on the particular sub-type. These fibers are quicker to fatigue.


    36: Fatty Acid Pathways and Regulation (Worksheet) is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by LibreTexts.

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