Bioc 460 Spring 2008 - Lecture 36 (Miesfeld)

1. Lipid Metabolism 2:Acetyl-CoA carboxylase, fatty acid synthase reaction, and regulation of fatty acid synthesis C247 Bioc 460 Spring 2008 - Lecture 36 (Miesfeld) The fatty acid synthase enzyme in eukaryotes is dimer of two very large polypeptide chains, each encoding seven functional units C247 is a fatty acid synthase inhibitor that reduces breast cancer incidence in mice AMP-activated kinase (AMPK) is a regulator of acetyl-CoA carboxylase

2. Key Concepts in Lipid Metabolism • Fatty acid synthesis takes place in the cytosol, uses NADPH as coenzyme in redox reactions, and the building block is malonyl-CoA. • Acetyl-CoA carboxylase is the key regulated enzyme in fatty acid synthesis and is responsible for generating malonyl-CoA in a carboxylation reaction using acetyl-CoA. • The fatty acid synthase protein complex consists of six enzymatic activities and the acyl carrier protein; each reaction cycle adds two carbons that are derived from malonyl-CoA following decarboxylation. • The citrate shuttle is responsible for moving acetyl-CoA equivalents from the mitochondrial matrix to the cytosol.

3. While the chemistry of the four core reactions required for the removal or addition of C2 acetyl groups to the hydrocarbon chain are similar between fatty acid degradation and synthesis, the two pathways are in fact quite distinct in terms of the required enzymes, subcellular location and source of redox energy. Comparison of fatty acid synthesis and degradation

4. Review of Pathway Questions 1. What purpose does fatty acid synthesis serve in animals? Fatty acid synthesis reactions in the cytosol of liver and adipose cells convert excess acetyl CoA that builds up in the mitochondrial matrix when glucose levels are high into fatty acids that can be stored or exported as triacylglycerols. 2. What is the net reaction in the synthesis C16 palmitate? Fatty acid synthesis:8 Acetyl CoA + 7 ATP + 14 NADPH + 14 H+→ Palmitate + 8 CoA + 7 ADP + 7 Pi + 14 NADP+ + 6 H2O

5. Review of Pathway Questions 3. What are the key enzymes in fatty acid synthesis?Acetyl CoA carboxylase - catalyzes the commitment step in fatty acid synthesis using a biotin-mediated reaction mechanism that carboxylates acetyl-CoA to form the C3 compound malonyl-CoA.. Fatty acid synthase - this large multi-functional enzyme is responsible for catalyzing a series of reactions that sequentially adds C2 units to a growing fatty acid chain covalently attached to the enzyme complex. 4. What is a real life application of fatty acid synthesis?

6. Acetyl-CoA carboxylase catalyzes the commitment step in fatty acid synthesis by converting acetyl-CoA to malonyl-CoA C2 acetate unit Is the energy charge high or low in the cell when excess acetyl-CoA is available for fatty acid synthesis?

7. The swinging arm mechanism of acetyl-CoA carboxylase

8. The fatty synthesis reaction cycle ACP Acetyl-CoA is the priming group only in the first cycle, after that, only malonyl-CoA is added to the ACP carrier protein each time. There are four reaction steps required each cycle to result in the net addition two carbons to the growing fatty acid chain. ACP ACP ACP ACP

9. The fatty synthesis reaction cycle Each cycle of the fatty acid synthase reaction requires the input of one malonyl-CoA and the oxidation of 2 NADPH molecules (4 e- total). The synthesis of C16 palmitate therefore requires 14 NADPH.

10. The fatty synthesis reaction cycle In the final step, the enzyme palmitoyl thioesterase catalyzes a hydrolysis reaction to release palmitate.

11. The fatty synthesis reaction cycle Acetyl-CoA is added first to the ACP and then transferred to the KS subunit (not shown here).

12. The “ready to go” chain is then moved back to KS to leave the ACP protein open for a new malonyl-CoA. The acetyl-CoA is only added in the 1st round. The chemical reactions take place on the ACP protein.

13. Summary of the fatty synthesis pathway • We begin by forming seven molecules of malonyl CoA using the acetyl CoA carboxylase reaction: • 7 Acetyl CoA + 7 CO2 + 7 ATP --> 7 malonyl + 7 ADP + 7 Pi • We then use these seven malonyl CoA molecules for seven turns of the reaction cycle beginning with the priming of fatty acid synthase by one molecule of acetyl CoA: • 1 Acetyl CoA + 7 malonyl CoA + 14 NADPH + 14 H+ --> • palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O There are 7 dehydration steps required for palmitate synthesis, why only 6 net H2O?

14. Summary of the fatty synthesis pathway • The net fatty acid synthesis reaction for palmitate (C18) can then be written as: • 8 Acetyl CoA + 7 ATP + 14 NADPH + 14 H+ --> • palmitate + 8 CoA + 7 ADP + 7 Pi + 14 NADP+ + 6 H2O Write the net reaction for the synthesis of C18 stearate.

15. The citrate shuttle transports acetyl-CoA equivalents from the matrix to the cytosol and generates NADPH The citrate uses malic enzyme to generate 1 NADPH for each acetyl-CoA equivalent that is transported. The bulk of NADPH needed for fatty acid synthesis comes from the PPP. Why does this makes sense in terms of glucose-6P levels?

17. Regulation of fatty acid synthesis Acetyl CoA carboxylase is most active when it is in a homopolymeric form. Citrate and palmitoyl CoA are metabolites that bind to an allosteric site on the enzyme stimulating polymerization or depolymerization, respectively.

18. Metabolic regulation of acetyl-CoA carboxylase

19. Hormonal regulation of acetyl-CoA carboxylase

20. AMPK is an important metabolic sensor

21. Three Metabolic Control Points of FA Synthesis What is the likely metabolic fate of the palmitoyl-CoA if this were a liver cell? What if it were a fat cell?