fatty acid synthesis + citrate-malate shuttle+ regulation (Slide deck 2)

steps and structures

differences between fatty acid breakdown and biosynthesis

• Occur by different pathways

  • Biosynthesis requires malonyl-CoA

• Catalyzed by different sets of enzymes

• Occur in different cellular compartments (in eukaryotes)

  • Breakdown occurs in the mitochondria

  • Biosynthesis occurs in the cytosol

fat synthesis: step 1

malonyl-CoA is formed from acetyl-CoA and bicarbonate

• acetyl-CoA carboxylase = catalyzes the irreversible formation of malonyl-CoA from acetyl-CoA

  • Reaction occurs in the cytoplasm

  • Contains a biotin prosthetic group covalently bound in amide linkage to the 𝜀-amino group of a Lys residue

• Step 1: the carboxyl group from HCO₃^- is transferred to biotin in an ATP-dependent reaction

  • The carboxyl group is carried by the biotin to a different active site, where the CO₂ is transferred to acetyl-CoA to yield malonyl-CoA. This functions to make the subsequent steps more thermodynamically favorable.

why is bicarbonate used to activate acetyl-CoA

• It's a good leaving group once attached

• It’s around → forms spontaneously when carbon dioxide dissolves in water 

• Biology is complex and doesn’t always use the most straightforward pathway

fatty acid synthesis: step 2 

begins with malonyl-ACP and Acetyl group (from acetyl-CoA) at the KS domain

condensation reaction, CO₂ lost allows it to be favorable

• catalyzed by fatty acid synthase I

• forming beta-ketoacyl-ACP

how many carbons does the chain elongate by?

2

fatty acid synthesis: step 3

reduction reaction with NADPH in cytoplasm

• doesn’t cost any ATP

• catalyzed by fatty acid synthase I

• forming beta-hydroxyacyl-ACP

fatty acid synthesis: step 4

dehydration reaction which removes H₂O

• makes a double bond

• catalyzed by fatty acid synthase I

• forming enoyl-ACP

fatty acid synthesis: step 5

reduction reaction using NADPH

• again doesn’t cost any energy

• catalyzed by fatty acid synthase I

• forms butaryl-ACP

fatty acid synthesis: step 6

translocation step so chain can keep growing

• rearranges and puts the thiol group on the ACP arm and the Butaryl on the KS domain

• catalyzed by fatty acid synthase I

• then the steps repeat to elongate chain again 

  • a new incoming malonyl can be added until the FA is 16 carbons long

flexibility of ACP and KS domains

ACP arm is flexible but KS (β-ketoacyl synthase, where acetyl-CoA will bind, thiol linkage) is not flexible

difference between cofactor and activating groups in fatty acid breakdown and biosynthesis

• In 𝛽 oxidation:

  • NAD and FAD serve as electron acceptors

  • The activating group is the thiol (-SH) group of coenzyme A

• In fatty acid synthesis:

  • The reducing agent is NADPH

  • The activating groups are two different enzyme-bound -SH groups

fatty acid synthase 1 (FAS I) —> structure explanation

• FAS I is found in mammals

• Seven active sites are in separate domains within a single multifunctional polypeptide chain

  • The intermediates remain covalently attached as thioesters to one of two thiol groups:

    • The -SH group of a Cys residue in 𝛽-ketoacyl-ACP synthase (KS)

    • The -SH group of acyl carrier protein (ACP)

• Two polypeptide chains function independently, but as a homodimer 

  • Homodimer: The enzyme is made of two identical polypeptide chains (subunits) that come together to form the functional complex

  • One chain can carry out fatty acid synthesis on its own → that’s what “function independently” means.

  • Two of these chains naturally dimerize (stick together in a head-to-tail orientation).

  • As a homodimer, they work more efficiently — the growing fatty acid chain can even “swing” between the two monomers during synthesis.

• ACP arm within protein can go to reaction centers since it is flexible

how do substrates within the fatty acid synthase I know where to go?

• Substrates know which site to go to based on attraction and lock and key

  • Each catalytic domain has a specific binding pocket with the right shape and chemical attractions

  • The flexible ACP arm increases efficiency because it can reach multiple domains, but the chemical recognition ensures it doesn’t “dock” at the wrong place.

why is the dimer organization of fatty acid synthase I imporant?

The dimeric organization makes the process more tolerant of mistakes, because there’s a “backup” active site and the intermediates can switch over

The overall process of palmitate synthesis ** might have carbon tracking question

Carbons C-16 and C-15 of the palmitate are derived from the methyl and carboxyl carbon atoms, respectively, of an acetyl-CoA used to prime the system at the outset

Acyl carrier protein (ACP)

• Contains 4’-phosphopantetheine

• 4’-phosphopantetheine = a prosthetic group of ACP that serves as a flexible arm

  • Also in coenzyme A

  • Tethers the fatty acyl chain to the surface of the FAS complex

  • Carries reaction intermediates from one active site to the next

Acyl carrier protein (ACP) and coenzyme A

• Incredibly similar structure

• And are great leaving groups

• The acyl carrier protein (ACP) itself is not a leaving group; instead, it carries an acyl group attached to its phosphopantetheine (PPT) arm via a thioester bond

biosynthesis of fatty acid requires (3)

• Acetyl-CoA

• The group transfer potential of ATP to make malonyl-CoA

• The reducing power of NADPH to reduce the 𝛽-keto group and the double bond

citrate-malate shuttle

• We need to get the acetyl-CoA which is required to make a lipid into the cytoplasm

• Acetyl-CoA is produced in the mitochondrial matrix by pyruvate dehydrogenase -- but acetyl-CoA cannot diffuse through the mitochondrial membrane to the cytoplasm

• TCA/Kreb’s cycle converts acetyl-CoA into citrate and there IS a transporter for citrate

• Citrate in the cytoplasm is converted into acetyl-CoA by citrate lyase → for fatty acid synthesis

• But now need to restore the mitochondrial stores of citrate

  • The product of citrate lyase is oxaloacetate, which after conversion to malate, can diffuse through the outer mitochondrial membrane and be transported back into the matrix

  • OR: malate can be converted to pyruvate by malic enzyme which is transported into the matrix by the pyruvate transporter and converted into oxaloacetate. This pathway involving malic enzyme produces NADPH -- which is required for fatty acid syntehsis and is one of the major ways in which the cell generates this essential electron acceptor. 

• The return of oxaloacetate back into the mitochdonrial matrix is facilitated by one of these two transporters:

  • Malate-𝛼-ketogluterate transporter = transports malate into the matrix where it is reoxidized to oxaloacetate by malate dehydrogenase

  • Pyruvate transporter = transports pyruvate into the matrix where it is converted to oxaloacetate by pyruvate carboxylase

two main ways of generating cytosolic NADPH

• pentose phosphate pathway

• malic enzyme (citrate-malate shuttle)

acetyl-CoA carboxylase (ACC) regulation

• We want to synthesize fatty acids when there is an abundance of energy and acetyl-CoA available 

• We want to reduce/restrict synthesis when there is not

• acetyl-CoA carboxylase -- which synthesizes malonyl-CoA (a required substrate for FA synthesis) is inhibited by glucagon (secreted when blood glucose levels are low in the fasting state) and by high levels of palmitoyl-CoA the ultimate product of fatty acid synthase (FAS)

• Negatively regulated by phosphorylation

ACC and CAT1 regulation

• ACC -- which makes malonyl-CoA is inhibited when phosphorylated

• High blood glucose dephosphorylates this enzyme making it more active

• The product, malonyl-CoA inhibits carnitine acyl-transferase I. Restricting the amount og fatty acyl-CoA that can enter the mitochondria for oxidative breakdown

• In the fasting state ACC is inactive and malonyl CoA is not being synthesized and beta-oxidation is favoured.