Pentose Phosphate Pathway Nitrogen, Sulfur & Amino Acids LECTURES-OUTLINE Introduction Overview Glycolysis Fermentation TCA cycle Respiration Gluconeogenesis Glycogen Photosystems Fatty acids Pentose Phosphate Pathway Nitrogen, Sulfur & Amino Acids Integration (Eating and exercise) Lipids Nucleotides BICH411/ M. Polymenis

METABOLISM SIMPLIFIED ADP + Pi ATP Reduced biosynthetic products Oxidized precursors ANABOLISM NADP+ NADPH + H+ Reduced fuel Oxidized products CATABOLISM NAD+ NADH + H+ OXPHOS H2O O2 ATP ADP + Pi [Adapted from Biochemistry (2nd ed), Wood et al (1981)] BICH411/ M. Polymenis

METABOLISM SIMPLIFIED Fatty acids NADP+ NADPH + H+ Oxidized products CATABOLISM Breakdown of larger molecules Sugars NAD+ NADH + H+ Amino Acids [Adapted from Biochemistry (2nd ed), Wood et al (1981)] BICH411/ M. Polymenis

METABOLISM SIMPLIFIED Fatty acids NADP+ NADPH + H+ Oxidized products Glucose CATABOLISM Breakdown of larger molecules NAD+ NADH + H+ Amino Acids [Adapted from Biochemistry (2nd ed), Wood et al (1981)] BICH411/ M. Polymenis

FATTY ACIDS Overview Mobilization (fat cells and diet) Oxidation/breakdown Some variations in breakdown Ketogenesis BICH411/ M. Polymenis

FATTY ACIDS Carboxylic acids consisting of a hydrocarbon chain and a terminal carboxyl group BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS Why are they such good energy ‘reserves’? BICH411/ M. Polymenis

FATTY ACIDS Reduced ‘fuel’ Caproic (hexanoic) acid Caprylic (octanoic) acid BICH411/ M. Polymenis

FATTY ACIDS Caproic (hexanoic) acid Hexane Hexose Caprylic (octanoic) acid Octane BICH411/ M. Polymenis

FATTY ACIDS Why are they such good energy ‘reserves’? Reduced Less hydrated => occupy less space BICH411/ M. Polymenis

FATTY ACIDS Saturated: No double bonds (‘saturated’ with as many H atoms as possible) BICH411/ M. Polymenis

FATTY ACIDS Saturated: No double bonds (‘saturated’ with as many H atoms as possible) Unsaturated: ≥ 1 double bond Mono-unsaturated: 1 double bond Poly-unsaturated: >1 BICH411/ M. Polymenis

FATTY ACIDS Stearic acid (saturated) in animal fat (~30%) [18:0] Elaidic acid (mono-unsaturated) trans in vegetable oils [18:1 trans-9] Oleic acid (mono-unsaturated cis In olive oil (55-80%) [18:1 cis-9] BICH411/ M. Polymenis

FATTY ACIDS Overview Mobilization (fat cells and diet) BICH411/ M. Polymenis

FATTY ACIDS Triacylglycerols (triglycerides): Esters of glycerol and fatty acids BICH411/ M. Polymenis

FATTY ACIDS (Image from Harvard School of Public Health) BICH411/ M. Polymenis

FATTY ACIDS FAT MOBILIZATION (From biowiki.ucdavis.edu) BICH411/ M. Polymenis

FATTY ACIDS DIETARY FAT Pancreatic lipase generates monoacylglycerols and free fatty acids BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS What happens to glycerol after triglyceride breakdown by lipases? BICH411/ M. Polymenis

GLUCONEOGENESIS ATP GLYCEROL KINASE Glycerol Glycerol-3-P GLYCEROL-3-P DEHYDROGENASE 2H DHAP IN THE LIVER BICH411/ M. Polymenis

FATTY ACIDS Human Serum Albumin BICH411/ M. Polymenis

FATTY ACIDS Serum Albumin >50% of protein in the serum Binds and transports Drugs Hormones Fatty acids Maintains osmotic balance pH BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS Overview Mobilization (fat cells and diet) Oxidation/breakdown BICH411/ M. Polymenis

FATTY ACIDS Fatty acids are broken down in 2C units -Franz Knoop, 1904 BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

H3C(CH2)14-COO + 8CoASH → 8 acetyl-CoA FATTY ACIDS Fatty acid breakdown yields acetyl-CoA  TCA cycle input H3C(CH2)14-COO + 8CoASH → 8 acetyl-CoA (palmitate) BICH411/ M. Polymenis

FATTY ACIDS H3C(CH2)12-CH2-CH2-COO → β α → H3C(CH2)12-COO + H3C-CO-X β α → H3C(CH2)12-COO + H3C-CO-X β α BICH411/ M. Polymenis

FATTY ACIDS H3C(CH2)12-CH2-CH2-COO → β α → H3C(CH2)12-COO + H3C-CO-X β α → H3C(CH2)12-COO + H3C-CO-X β α 2 chemical problems: Oxidize the β-carbon to a β-keto group Stabilize the α-carbanion that will be produced at the C-C bond cleavage BICH411/ M. Polymenis

FATTY ACIDS H3C(CH2)12-CH2-CH2-COO → β α → H3C(CH2)12-COO + H3C-CO-X β α → H3C(CH2)12-COO + H3C-CO-X β α 2 chemical problems: Oxidize the β-carbon to a β-keto group Stabilize the α-carbanion that will be produced at the C-C bond cleavage BICH411/ M. Polymenis

FATTY ACIDS O- O | ↔ || H2C=C-SCoA -CH2-C-SCoA BICH411/ M. Polymenis

FATTY ACIDS RCOO- + CoASH +ATP → RCO-SCoA + AMP + PPi Acyl-CoA SYNTHETASE BICH411/ M. Polymenis

FATTY ACIDS RCOO- + CoASH +ATP → RCO-SCoA + AMP + PPi Acyl-CoA SYNTHETASE BICH411/ M. Polymenis

FATTY ACIDS RCOO- → RCO-SCoA ACTIVATION Acyl-CoA SYNTHETASE BICH411/ M. Polymenis

Another spatial problem FATTY ACIDS ACTIVATION RCOO- → RCO-SCoA Acyl-CoA SYNTHETASE CYTOSOL BREAKDOWN MITOCHONDRIA Another spatial problem BICH411/ M. Polymenis

FATTY ACIDS (Image from WikiMedia) BICH411/ M. Polymenis

FATTY ACIDS Carnitine (from Lys and Met) (Image from WikiMedia) BICH411/ M. Polymenis

RATE-LIMITING FOR FATTY ACID OXIDATION FATTY ACIDS RATE-LIMITING FOR FATTY ACID OXIDATION (Image from WikiMedia) BICH411/ M. Polymenis

FATTY ACIDS RCOO- → RCO-SCoA(cyt) RCO-SCoA(mit) ACTIVATION TRANSPORT Acyl-CoA SYNTHETASE CARNITINE ACYLTRANSFERASE SHUTTLE BICH411/ M. Polymenis

H3C(CH2)12-CH2-CH2-COSCoA + CoASH → FATTY ACIDS H3C(CH2)12-CH2-CH2-COSCoA + CoASH → β α → H3C(CH2)12-COSCoA + H3C-COSCoA β α 2 chemical problems: Oxidize the β-carbon to a β-keto group Stabilize the α-carbanion that will be produced at the C-C bond cleavage BICH411/ M. Polymenis

FATTY ACIDS The 4 steps of -oxidation BICH411/ M. Polymenis

FATTY ACIDS -carbon BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS RCOO- → RCO-SCoA(cyt) RCO-SCoA(mit) Acetyl-CoA + R’CO-SCoA ACTIVATION TRANSPORT RCOO- → RCO-SCoA(cyt) RCO-SCoA(mit) Acyl-CoA SYNTHETASE CARNITINE ACYLTRANSFERASE SHUTTLE OXIDATION Acetyl-CoA + R’CO-SCoA BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS ATP yield per cycle 1 FADH2 (~1.5 ATP) 1 NADH2 (~2.5 ATP) 1 Acetyl-CoA (~10ATP) ________ ~14 ATP For a c2n fatty acid: n-1 oxidation cycles needed 1 Acetyl-CoA (10 ATP) 2 ATP were used in activation For palmitate (c16, n=8): (8-1)x14 + 10 – 2 = 106 BICH411/ M. Polymenis

FATTY ACIDS Caproic (hexanoic) acid BICH411/ M. Polymenis

FATTY ACIDS Caproic (hexanoic) acid (c6, n=3): (3-1)x14 + 10 – 2 = 36 ATP BICH411/ M. Polymenis

FATTY ACIDS Caproic (hexanoic) acid (c6, n=3): (3-1)x14 + 10 – 2 = 36 ATP 32 ATP Glucose BICH411/ M. Polymenis

FATTY ACIDS FA length ATP yield 6 36 12 78 18 120 24 162 6 36 12 78 18 120 24 162 BICH411/ M. Polymenis

FATTY ACIDS FA length ATP yield 6 32 12 78 18 120 24 162 6 32 12 78 18 120 24 162 LENGTH MATTERS BICH411/ M. Polymenis

FATTY ACIDS Why are they such good energy ‘reserves’? Reduced Less hydrated => occupy less space BICH411/ M. Polymenis

ATP SYNTHASE Pi +ADP ↔ H2O + ATP ATP ATP Pi ADP H2O Energy Open Tight Loose Pi +ADP ↔ H2O + ATP ATP ATP Pi ADP H2O BICH411/ M. Polymenis

ATP SYNTHASE Pi +ADP ↔ H2O + ATP ATP ATP Pi ADP H2O Energy Open Tight Loose Pi +ADP ↔ H2O + ATP ATP ATP Pi ADP H2O BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS Robert A. Egnatchik & Ralph J. DeBerardinis Nature (2015) doi:10.1038/nature14375 BICH411/ M. Polymenis

FATTY ACIDS Overview Mobilization (fat cells and diet) Oxidation/breakdown Some variations in breakdown BICH411/ M. Polymenis

FATTY ACIDS Very long fatty acids (> c20-22), they first undergo β-oxidation in peroxisomes No ATP synthase: electrons are transferred to oxygen → hydrogen peroxide Different carnitine shuttle BICH411/ M. Polymenis

FATTY ACIDS What about odd-carbon fatty acids? End up with propionyl- , not acetyl-CoA CH3CH2CO-SCoA vs. CH3CO-SCoA BICH411/ M. Polymenis

FATTY ACIDS What about odd-carbon fatty acids? End up with propionyl- , not acetyl-CoA CH3CH2CO-SCoA vs. CH3CO-SCoA Succinyl-CoA BICH411/ M. Polymenis

GLUCONEOGENESIS In animals …. gluconeogenesis [pyruvate, TCA cycle intermediates] glucose gluconeogenesis Fat [Acetyl-CoA] glucose BICH411/ M. Polymenis

FATTY ACIDS What about odd-carbon fatty acids? End up with propionyl- , not acetyl-CoA CH3CH2CO-SCoA vs. CH3CO-SCoA Succinyl-CoA GLUCONEOGENESIS BICH411/ M. Polymenis

FATTY ACIDS What about odd-carbon fatty acids? End up with propionyl- , not acetyl-CoA CH3CH2CO-SCoA vs. CH3CO-SCoA Succinyl-CoA TCA CYCLE GLUCONEOGENESIS BICH411/ M. Polymenis

FATTY ACIDS What about branched fatty acids? BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS What about unsaturated fatty acids? BICH411/ M. Polymenis

FATTY ACIDS What about unsaturated fatty acids? Move the double bond between the ,  carbons BICH411/ M. Polymenis

FATTY ACIDS What about unsaturated fatty acids? ENOYL-COA-ISOMERASE BICH411/ M. Polymenis

FATTY ACIDS What about unsaturated fatty acids? Move the double bond between the ,  carbons Then simply feed it to Enoyl-CoA hydratase BYPASS OF THE 1ST (FADH2 –GENERATING) REACTION BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS Overview Mobilization (fat cells and diet) Oxidation/breakdown Some variations in breakdown Ketogenesis BICH411/ M. Polymenis

FATTY ACIDS WHAT: After fatty acid breakdown, some of the acetyl-CoA is used to make ketone bodies (acetone, acetoacetate, β-hydroxybutyrate) WHERE: Mitochondria (liver) WHY: Very ‘portable’ form of fatty acid BICH411/ M. Polymenis

FATTY ACIDS WHEN: Carbohydrates LOW → Gluconeogenesis UP → TCA intermediates (oxaloaceate) → LOW BICH411/ M. Polymenis

FATTY ACIDS WHEN: Carbohydrates LOW → Gluconeogenesis UP → TCA intermediates (oxaloaceate) → LOW Carbohydrates LOW → Fatty acid breakdown UP → Acetyl-CoA UP BICH411/ M. Polymenis

FATTY ACIDS WHEN: Carbohydrates LOW → Gluconeogenesis UP → TCA intermediates (oxaloaceate) → LOW Carbohydrates LOW → Fatty acid breakdown UP → Acetyl-CoA UP → Ketogenesis BICH411/ M. Polymenis

FATTY ACIDS HOW? BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS Ac-CoA an electrophile Ac-CoA a nucleophile BICH411/ M. Polymenis

FATTY ACIDS AcCoA: Chemical versatility explains its central metabolic role AcCoA an electrophile AcCoA a nucleophile Acylthioesters is the general “currency” of acyl-transfer processes BICH411/ M. Polymenis

FATTY ACIDS HOW? BICH411/ M. Polymenis

FATTY ACIDS The mechanism of HMG-CoA synthase has been elucidated by enzyme intermediate structures of the acetylated-enzyme AcAc-CoA complex (blue) and the covalent HMG-CoA-enzyme complex (purple) (7). The overall reaction of acetyl-CoA and AcAc-CoA to produce HMG-CoA is depicted in three phases. (A) Acetylation/deacetylation. The substrates bind to the enzyme with the acetyl-CoA, forming a covalent acetylated-cysteine intermediate. (B) Condensation/cleavage. Upon dissociation of the CoASH, the second substrate AcAc-CoA enters the active site, forming the acetylated-enzyme AcAc-CoA complex shown in blue. The condensation reaction is shown going through an enolate intermediate to form the covalent HMG-CoA-enzyme complex shown in purple. (C) Hydrolysis/dehydration. The final phase of the overall reaction is the hydrolysis of the covalent HMG-CoA-enzyme complex, which occurs through a tetrahedral intermediate to form HMG-CoA as the sole product of the reaction. The chemically challenging step for this reaction is the condensation step that features the formation of a carbon–carbon bond. Bahnson B J PNAS 2004;101:16399-16400 ©2004 by National Academy of Sciences

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS BICH411/ M. Polymenis

FATTY ACIDS Overview Mobilization (fat cells and diet) Oxidation/breakdown Some variations in breakdown Ketogenesis BICH411/ M. Polymenis