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G6P  can be oxidized to ribulose 5 phosphate (for PPP) - Glycogenesis, glycogenolysis, gluconeogenesis, glycolysisOxidation-Reduction Coenzymes NAD  cofactor for substrate oxidation (dehydrogenases) NADPH  cofactor for reductases (substrate reduction)Activation-Transfer Coenzymes - Participate di...

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G6P  can be oxidized to ribulose 5 phosphate (for PPP) - Glycogenesis, glycogenolysis, gluconeogenesis, glycolysis Oxidation-Reduction Coenzymes NAD  cofactor for substrate oxidation (dehydrogenases) NADPH  cofactor for reductases (substrate reduction) Activation-Transfer Coenzymes - Participate directly in catalysis by forming covalent bond with portion of substrate - Tightly held moiety activated for transfer, addition of water or some other reaction - Portion of coenzyme that forms covalent bond with substrate  functional group - eg. TPP, CoA, biotin and pyrodixal phosphate  synthesized from vitamins - TPP (B1): PDH, a-ketoglutarate, transketolase… - PLP (B6): Schiff base linkage (glycogen phosphorylase in glycogenolysis, aminotransferases in deamination of amino acids) - Biotin (B7) usually used for carboxylations – eg. ACC in FA biosynthesis, pyruvate carboxylase Transaldolase uses lysine side chain

Glycolysis 1. Energy Investment phase a. Glucose  G6P (ATP used)  Fructose-6-P  Fructose 1,6-P (PFK-1 – ATP used)  G3P and DHAP 2. Energy payoff phase a. G3P  1,3 bisphosphoglycerate (ATP produced)  3 phosphoglycerate  2 phosphoglycerate  PEP  pyruvate (x2) Glucose-6-phosphate  glycolysis, PPP, glycogen synthesis or other pathways NADH transferred to ETC through malate-aspartate shuttle and glycerol-3-phosphate shuttle Malate aspartate shuttle: OAA reduced to malate, transferred to inside of mitochondria, oxidized to OAA  aspartate by aminotransferase transferred to outside  OAA G3P Shuttle: Electrons from matrix of mitochondria to ETC NADH oxidized  electrons reduce DHAP to G3P, G3P oxidized to DHAP  reduces FAD in ETC Pyruvate  acetyl coA by Pyruvate DH with cofactor TPP, lipoate, FAD (oxidative decarboxylation) Net per mol of glucose: 2ATP, 2NADH, 2 pyruvate 30-32 ATP for aerobic 3 negative free energy changes catalysed by: hexokinase, PFK, pyruvate kinase Hexokinase and PFK-1: major regulatory enzymes in skeletal muscle Regulation of Pyruvate Kinase only in liver

PFK activated by AMP and fructose,2,6 bp and AMP PFK inhibited by ATP and citrate Insulin activates glucokinase, PFK-1 and pyruvate kinase Glucagon inhibits Regulatory Enzymes of TCA cycle: citrate synthase, a-ketoglutarate DH, isocitrate dehydrogenase Anaerobic Conversion of Pyruvate Pyruvate  lactate by lactate DH Yeast cells convert lactate to ethanol Pyruvate  acetaldehyde  ethanol (decarboxylation and reduction) – uses TPP and alcohol DH Produces 2 ATP Pyruvate to Acetyl CoA (Oxidative Decarboxylation) - PDH complex - E1, E2, E3 - Using cofactors TPP, lipoate and FAD - Entry of CoA-SH, reduction of NAD 1. Pyruvate reacts with TPP (vitamin B1)  decarboxylation to form hydroxethyl-TPP of E1 2. Aldehyde group transferred to lipoamide cofactor of E2 and oxidized to acetyl 3. Acetyl transferred to next lipoamide cofactor (sulfur) 4. Transferred to coA-SH forming acetyl-coA. Product is released 5. E3 oxidises lipoamide by transferring 2 H atoms to cofactor FAD 6. FADH2 oxidised to FAD by NAD and enzyme complex ready for next cycle Pentose Phosphate Pathway Also known as hexose monophosphate shunt Oxidative portion: Glucose-6-phosphate  ribulose-5-phosphate by G6P dehydrogenase Generates ribose-5-phosphate for nucleotide synthesis and NADPH for reducing power Non oxidative portion Transform C5 to C7, C3, C4, etc using transketolases and transaldolases Net result of 3 mol ribulose 5 phosphate  2 mol fructose 6-P and 1 mole of glyceraldehyde 3-P which can continue through glycolytic pathway

Interchange of groups on single carbon atom: epimerization  produces xylulose 5phosphate Interchange of groups between carbon atoms: isomerization  produces ribose 5 phosphate Transketolase: transfer of 2C group from ketose to aldose using TPP (abstraction of acidic thiazole proton) – formation of 7C and 6C (fructose 6 P) Transaldolase: Transfers 3C fragment from sedoheptulose-7P to G3P  erythrose-4P and fructose 6P – uses active amino group on side chain of lysine to catalyse Glyoxylate Pathway Fats  carbs (acetyl coA from fat to glucose) In seedlings. Bypass decarboxylation steps for net synthesis of OAA (use fats for easier dispersion – lighter in weight and produce twice as much energy as carbs) Unique enzymes: isocitrate lyase and malate synthase Citrate  isocitrate  succinate + glyoxylate (by isocitrate lyase)  acetyl coA  malate (by malate synthase)  OAA … Succinate can be converted to malate in mitochondrion for use in gluconeogenesis or TCA Pyruvate  OAA (Gluconeogenesis) - By pyruvate carboxylase o 2 step mechanism: o ATP dependent carboxylation of cofactor to give N-carboxybiotin o Activated derivative transfers carboxyl to pyruvate o Uses biotin as cofactor, linked to e-amino group of lysine residue which swings to transfer carboxyl o HCO3- enters Glycolysis vs Gluconeogenesis in Liver - Glycolysis: Catabolic o Pyruvate kinase: PEP  Pyruvate - Gluconeogenesis: Reverse glycolysis o Lactate, alanine, amino acids (from TCA), glycerol  pyruvate o PEP carboxykinase: OAA  PEP o Pyruvate  OAA (by pyruvate carboxylase which requires biotin – compartmentalized reaction, use malate-aspartate shuttle to move OAA to cytosol)  PEP  G3P  Fructose 1,6-P …. Steps that don’t use enzymes of glycolysis  irreversible, regulated steps of glycolysis 1. Pyruvate  PEP (acetyl coA stimulates pyruvate carboxylase) 2. Fructose-1,6 bisphosphate  Fructose 6 phosphate (stimulated by citrate, inhibited by fructose 2,6 bisphosphatase and AMP) 3. G6P  glucose by g6p phosphatase (present in ER, liver and kidney but absent from muscles and brain) uses important intermediate phosphohistidine Consumption of 6 nucleoside triphosphates drives process forward. Fructose conversion to C3 molecules - Metabolised by conversion to G3P and DHAP (intermediates of glycolysis)

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Fructose  Fructose 1-P by Fructokinase (high Vmax) Fructose 1-P cleaved by aldolase  DHAP and G3P  a. pyruvate b. TCA c. fatty acid synthesis or can be converted to glucose by d. gluconeogenesis Fate of fructose parallels that of glucose

Polyol Pathway - Fructose synthesis from glucose - Contributes to formation of cataracts - Large amounts of glucose present (diabetes mellitus)  hexokinase is saturated and excess glucose enters polyol pathway - Glucose  Sorbitol by Aldolase reductase - Sorbitol  Fructose by Sorbitol DH - Hexokinase returns molecule to glycolysis pathway phosphorylating fructose to form fructose-6-phosphate - Uncontrolled diabetics with high blood glucose – more than glycolysis pathway can handle. Reaction’s mass balance favors production of sorbitol  increased osmotic pressure in lens ETC Concept - Located in inner membrane of mitochondrion - Proteins existing as multiprotein complexes - Carriers alternate reduced and oxidized states as accept and donate electrons - Electrons drop in free energy as they go down chain and are finally passed to O2 forming H2O - Electrons are transferred from NADH or FADH2 - Passed through number of proteins including cytochromes to O2 - Generates no ATP directly - Breaks large free energy drop from food to O2 into smaller steps that release energy in manageable amounts NADH  Complex I  Complex II CoQ  Complex III  Cytochrome C  Complex IV  O2  ATP synthase (oxidative phosphorylation) Uncoupling of Oxidative Phosphorylation - DNP analog uncouples respiration from phosphorylation  rise in consumption of oxygen - Take proton and release on other side (matrix) - - eg. thermogenin: generates heat instead of ATP Basic Chemical Reactions - Photosynthetic reaction: oxidation reduction reaction o CO2 + H2O  CH2O + O2 (oxygenic) o Water is oxidized (reductant) and CO2 is reduced – oxygen evolved as by product -

Anoxygenic photosynthesis: H2O not the reductant o Green sulfur bacteria:  CO2 + H2S  CH2O + S  Hydrogen sulfide is the reductant

Photosynthesis Reactions - Overall process in 2 phases - Light reactions (thylakoid membrane) o H2O + NADP + ADP + Pi + n(hv)  O2 + ATP + NADPH - Dark reactions (stroma): o CO2 + NADPH + ATP  CH2O + NADP + ADP + Pi Linear electron flow: - Light  PS II  Pq  Cytochrome complex  Pc – PS I  Fd  NADP reductase  NADPH ATP synthase produces ATP by allowing protons to flow from matrix to stroma Stage 1: CO2 trapped as carboxylate and reduced to aldehyde-ketone level found in sugars Stage 2: Regeneration of acceptor molecule, RuBP Ribulose-1,5 bisphosphate + CO2  2 mols 3-phosphoglycerate (rubisco) (irreversible) Enediol intermediate formed 3-phosphoglycerate  1,3 bisphosphoglycerate (using ATP) 1,3 bisphosphoglycerate reduced to G3P (using NADPH) 6 rounds for hexose production  uses 12 ATP and 12 NADPH Lipid Metabolism Summary - TAGs  FAs  Acetyl coA (B-oxidation)  Ketone bodies or cholesterol or ATP - Acetyl coA  Fatty acids (FA synthesis)  TAGs Emulsification of TAGs by Bile Salts in Intestine 1. Cholic acid (bile acid) ionizes to give cognate bile salt 2. Hydrophobic surface of bile salt molecule associates with TAG and several complexes aggregate to form micelle 3. Hydrophilic faces outward allowing micelles to associate with pancreatic lipases 4. Hydrolytic action of lipases allow FAs to associate into much smaller micelle  absorbed through intestinal mucosa Fats mobilized from dietary intake and liver - Plasma lipoproteins: spherical macromolecular complexes of lipids and specific proteins (apolipoproteins/apoproteins). Include chylomycrons, VLDL, LDL and HDL Fate of chylomicrons - Synthesised in intestinal epithelial cells  lymph  blood: Chylomicrons dietary TG (FA transferred to muscle for use in B ox and transferred to adipose tissue for storage)  chylomicron remnants to liver Carnitine Cycle for Transport of Fatty Acyl CoAs into Mitochondria - Long chain fatty acyl coA derivatives converted to acylcarnitine derivatives

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Carnitine acyltransferase I (associated with outer mitochondrial membrane)  formation of O acylcarnitine which is transported across inner membrane by translocase Acylcarnitine passed to carnitine transferase II in matrix side of inner membrane  transfers fatty acyl group back to coA to reform fatty acyl coA leaving free carnitine which can return across membrane via translocase Fatty acyl coA  B oxidation

Activation to fatty acyl coA requires 2ATP B-oxidation pathway of fatty acyl coA (mitochondrial matrix) 1. Double bond introduced by enzyme catalyzed removal of 2 Hs from carbon 2 and 3. Coenzyme FAD needed (acyl-coA dehydrogenase) 2. Water adds to double bond to yield alcohol (enoyl-coA hydratase) 3. Alcohol group oxidized to ketone – coenzyme NAD used (B-hydrocyacyl-coA dehydrogenase) 4. Carbon-carbon bond broken to yield acetyl coA and chain shortened fatty acid (acylCoA acetyltransferase) Route of metabolism depends on length of FA (very long first shortened in peroxisome and then brought to mitochondria) Even number of carbons: Energy yield for one cycle of B-oxidation of palmitic acid: 17ATP Complete oxidation of Palmitic acid: 129 molecules ATP per molecule palmitate Number of acetyl coAs produced = half number of carbons in original FA Eg. Stearic acid has 18 carbons  9 acetyl coAs produced through 8 cycles Peroxisome Oxidation (for very long chain lengths) - One key difference: Instead of reducing ubiquinone in first step (FAD reduction), produces H2O2 Produces 15ATP from one cycle as FADH not generated from first step Odd number of carbons: - Final product: 3C propionyl coA Catalysed by biotin dependent propionyl coA carboxylase (initial carboxylation at a-carbon of propionyl coA to produce D-methylmalonyl coA Propionyl coA converted to Succinyl coA  TCA CYCLE Unsaturated Fatty Acids 2 additional enzymes: - Isomerase - Reductase Required to handle cis double bonds Eg. linoleic acid: B oxidation proceeds 3 cycles

FA Biosynthesis Fatty Acyl ACP  B-ketoacyl ACP (by malonyl coA)  3-D hydroxyacyl ACP  enoyl ACP  Fatty acyl ACP Acetate units commited to synthesis by formation of malonyl coA Carboxylation of acetyl coA to malonyl coA  irreversible and commited step of synthesis – Catalysed by acetyl coA carboxylase (ACC) which uses biotin - Citrate activates ACC, palmitoyl coA (final product) inhibits ACC - Elongation of FAs: Condensations of malonyl coA with acyl coA and NADPH associated reductions – addition of acetyl units Desaturases  formation of unsaturated fatty acids -

Control of FA synthesis: - Insulin promotes glucose uptake and dephosphorylation (activation) of pyruvate DH, citrate lyase, acetyl coA carboxylase (ACC) - AMP activated protein kinase and protein kinase A  inhibit ACC by phosphorylation - Citrate activates ACC - Malonyl coA inhibits acetyl carnitine transferase inhibiting transport into mitochondrial matrix Structure and Classification of Lipids 1. Simple esters (RCOOR’) 2. Triesters of glycerol  Triacylglycerols (neutral) and glycerophospholipids (contain charged phosphate groups) 3. Derivatives of sphingosine (amino alcohol)  sphingolipids  include sphingomyelins and glycolipids 4. Steroids 5. Based on 20 carbon acid  eicosanoids Pathways in Glycerophospholipid biosynthesis - G3P or DHAP (euk only) or diacylglycerol (euk only)  phosphatidic acid  TAGs or CDP diaglycerol  phospholipids (eg. phosphatidyl-inositol, phophatidylserine, phosphatidylglycerol …) Phosphatidic acid  CDP diacylglycerol using CTP - Phosphatidic acid is the central metabolite, derived from G3P, DHAP or diacylglycerol Ceramide – Precursor of Sphingolipids - Building block for all other sphingolipids Palmitoyl coA + Serine + reduction  sphinganine acylated to N-acylsphinganine (ceramide) -

Sphingomyelin: produced by transfer of phosphocholine from phosphatidylcholine Glycosylation of ceramide by sugar nucleotides  cerebrosides (galactosylceramide) which make up 15% of lipids of myelin sheath structures Cerebrosides that contain one or more sialic acid moieties  gangliosides

Eicosanoids - Arachidonic acid - Cyclooxygenation of arachidonic acid by prostaglandin H synthase - COX and Pox ACTIVITY - All prostaglandins are cyclopentanoid acids derived from arachidonic acid - Biosynthesis initiated by enzyme associated with ER called prostaglandin endoperoxide H synthase (PGHS) or cyclooxygenase COX - Enzyme that converts arachidonic acid to prostaglandin PGH2  2 distinct activities: o COX and gluthatione-dependent hydroxyperoxidase (POX) o PGHS reaction: H atom abstraction by tyrosine radical on enzyme followed by rearrangements to cyclize and incorporate 2 oxygen molecules o Reduction of peroxide at C15 completes reaction Inhibition of prostaglandin synthesis by aspirin  O-acetylates Ser30 on enzyme PGHS  anti-inflammatory effect Steroids - Isoprenoids - 4 fused rings: 3 six carbon rings and a five carbon D ring - Cholesterol  precursor for steroid hormones and bile salts Cholesterol Biosynthesis + Fates - Conversion of C2 fragments (acetate) to C6 isoprenoid precursor (mevalonate) - Conversion of C6 mevalonate to C30 squalene via c5 intermediates - Cyclisation of C30 squalene to C27 cholesterol Rate limiting step: Catalysed by HMG-CoA reductase. HMG CoA undergoes 2 reduction (NADPH dependent) to produce 3R mevalonate -

Biliary cholesterol Bile acids Cholesteryl esters

Fate of HDL: - Nascant HDL synthesized in liver and intestinal cells - Exchanges proteins with chylomicrons and VLDL - HDL picks up cholesterol from cell membranes – cholesterol converted to CE by LCAT - HDL transfers CE to VLDL in exchange for TG. CETP mediates exchange Returns excess cholesterol to liver Steroid hormones: - Desmolase converts cholesterol to pregnenolone – transported to ER where OH oxidation and migration of DB  progesterone Regulation of cholesterol synthesis: - HMG-CoA  mevalonate (RLS) by HMG-CoA reductase - Regulated covalently by PP2A, PKA and AMPKA - Glucagon  phosphorylation of HMG coA reductase (inactivation) - Insulin  dephosphorylation of HMG coA reductase (activation)

Ketogenesis - Formation of ketone bodies (easily transported) - Acetyl coA converted to 3 important ketone bodies: o Acetone o Acetoacetate o B-hydroxybutyrate -

Ketone bodies made mostly in liver (mitochondrial matrix) but important sourves of fuel and energy for many tissues (brain, heart, skeletal muscle) Easily transportable forms of fatty acids that move through circulatory system without need for complex formation with serum albumin and other FA binding proteins

2 Acetyl coA  acetoacetyl coA by thiolase Acetoacetyl coA  B-hydrocy-B-methylglutaryl-CoA (HMG-CoA) by HMG CoA synthase HMG-CoA  Acetoacetate by HMG-CoA lyase Acetoacetate  acetone and B hydroxybutyrate by B hydroxybutyrate dehydrogenase Acetyl coA: key intermediate between fat and carbohydrate metabolism Acetyl coA readily converted to fatty acids but cannot undergo net conversion to carbohydrate

Human RBCs: lack mitochondria  can’t use ketone bodies for energy, eyes and outer segment of retina. Rely on glucose conversion to lactate for ATP B-oxidation vs FA biosynthesis B oxidation: - In mitochondria - CoA: acyl group carrier - FAD is electron acceptor - L alcohol - NAD electron acceptor - C2 unit product is acetyl coA FA biosynthesis: - In cytoplasm - ACP is acyl group carrier - NADPH is electron donor - D alcohol - NADPH is electron donor - C2 unit donor is malonyl coA Amino acid catabolism: Fate of carbon atom Amino acids that form acetyl coA or acetoacetyl coA contribute to fatty acid and ketone bodies  ketogenic Amino acids that are degraded to pyruvate/TCA intermediates  Glucogenic

Some amino acids are both glucogenic and ketogenic

Amino acid catabolism: Fate of nitrogen atom Alanine (AA1) + a-ketoglutarate (amino group acceptor)  pyruvate + glutamate (amino acid 2) by Alanine aminotransferase (ALT) -

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Enzymes catalyzing these reactions: aminotransferase (transaminase) Coenzyme PLP used (derived from vitamin B6) – aldehyde group of coenzyme forms schiff base with lysine chain on enzyme  imine exchange. Stabilised by favourable bond between PLP and imine nitrogen No free ammonium released at any stage

Urea Cycle (mitochondria though 3 steps occur in cytosol) NH4+ HCO3  carbamoyl phosphate 1. CPS1 converts ammonia from glutamate into carbamoyl phosphate  combines with ornithine  citrulline by ornithine transcarbamoylase 2. Citruline + Aspartate  Arginosuccinate by arginosuccinate synthetase 3. Arginosuccinate cleaved  arginine and fumurate by arginosuccinate lyase 4. Guanidium group cleaved  ornithine and urea by arginase N-acetylglutamate activates CPSI Fumurate and aspartate  direct links to TCA cycle Fumurate  intermediate in TCA that can be converted to OAA Transamination can convert OAA to fumurate providing link between 2 reactions Synthesis of Amino Acids Amino acids can be synthesized from intermediates in glycolysis, PPP and TCA cycle Glutamate can be made from NH4+ and a-ketoglutarate by reductive amination (using NADH/NADPH) by glutamate DH Glutamate: major donor of amino groups, a-ketoglutarate: major acceptor of amino groups Phenylalanine  Tyrosine by Phenylalanine hydroxylase Phenylketonuria (PKU): Phenylalanine hydroxylase defective  not enough tyrosine produced  brain damage Nitrogenous Bases - Bases of nucleotides and nucleic acids  derivatives of either pyrimidine or purine - Pyrimidines: 6 membered heterocyclic aromstic rings containing 2 nitrogen atoms (G,C) - Purine: combination of pyrimidine ring with five membered imidazole ring to yield fused ring system (A,T/U) Adenosine: Nucleoside with physiological activity  autacoids (local hormone) Caffeine  alkaloid  blocks interaction of extracellular adenosine

Nucleotides are nucleoside phosphates Also carry chemical energy in the cell (eg. ATP, ADP, AMP, components of cofactors, metabolic control) Salvage Pathways for Synthesis of Pyrimidines and Purines - Catalyzed by adenosine phosphoribosyltransferase  free adenine + PRPP  adenine nucleotide (AMP) + PPi - Free guanine and hypoxanthine (deamination product of adenine) salvaged in same way by hypoxanthine guanine phosphoribosyltransferase - R...