Carbohydrates, Regulation of Metabolism

OUTLINE: Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Fermentations Pyruvate Krebs’ Cycle Oxidative Phosphorylation ANABOLISM I: Carbohydrates Carbon Assimilation – Calvin Cycle Exam-1 material Exam-2 material Electron transport Chemiosmotic theory: Phosphorylation Fat Catabolism Fatty acid Catabolism Exam-3 material Mobilization from tissues (mostly adipose) Activation of fatty acids Transport; carnitine Oxidation: b-oxidation, 4 steps: Protein Catabolism Amino-Acid Degradation Dealing with the nitrogen; Urea Cycle Dealing with the carbon; Seven Families Nucleic Acid & Nucleotide Degradation PHOTOSYNTHESIS: Overview of Photosynthesis Key experiments: Light Reactions energy in a photon pigments HOW Light absorbing complexes-”red-drop experiment” Reaction center Photosystems (PS) PSII – oxygen from water splitting PSI – NADPH Proton Motive Force – ATP Overview of light reactions Stage One – Rubisco Carboxylase Know mechanism Oxygenase Glycolate cycle Stage Two – making sugar Stage Three - remaking Ru 1,5P2 Overview and regulation Calvin cycle connections to biosynthesis Know pathway C4 versus C3 plants Kornberg cycle - glyoxylate Carbohydrate Biosynthesis in Animals precursors Cori cycle Know pathway reversible steps irreversible steps – four Gluconeogenesis energetics 2-steps to PEP in mitochondria: Pyr carboxylase-biotin & PEPCK FBPase G6Pase Glycogen Synthesis UDP-Glc Glycogen synthase branching Pentose-Phosphate Pathway oxidative-NADPH non-oxidative-Ribose 5-P Regulation of Carbohydrate Metabolism Acetyl-CoA/Pyruvate Biosynthesis of Lipids Pyruvate/PEP F6P/FBP: Fru 2,6P2 Glc/Glc6P: sequestration Glycogen: PKA/PP1 Insulin signaling Anaplerotic reactions Fatty Acids contrasts location & transport Synthesis End of Exam-4 material ANABOLISM I: Carbohydrates; Regulation of Metabolism Catabolism vs. Anabolism 1 Anaplerotic Reactions • Intermediates in the citric acid cycle can be used in biosynthetic pathways. • Must replenish the intermediates in order for the cycle and central metabolic pathway to continue. • In animals, these 4-carbon intermediates are formed by carboxylation of 3-carbon precursors. Aspartate + GTP AMP deaminase, adenylosuccinate Pyruvate carboxylase deficiency – an inherited metabolic disorder where anaplerosis is greatly reduced. – What is the problem? – How to treat this disorder? – Other anaplerotic substrates such as the odd-carbon-containing triglyceride triheptanoin are used NH4+ + GDP + Pi + Fumarate synthetase, adenylosuccinate lyase Enzyme Kornberg discovered Muscle ANABOLISM I: Summary What we learned: • Gluconeogenesis, a process by which cells can use a variety of metabolites for the synthesis of glucose • The differences between glycolysis and gluconeogenesis – how they are both made thermodynamically favorable – how they are differentially regulated to avoid a futile cycle • The pentose phosphate pathway, a process by which cells can generate pentose phosphates and NADPH. The pentose phosphates can be regenerated into glucose 6-phosphate, for which NO ATP is required. • living organisms regulate the flux of metabolites through metabolic pathways by: – increasing or decreasing enzyme concentrations – activating or inactivating key enzymes in the pathway • the activity of key enzymes in glycolysis and gluconeogenesis is tightly and coordinately regulated via various activating and inhibiting metabolites (Fru 2,6P2) • glycogen synthesis and degradation is regulated by hormones insulin, epinephrine, and glucagon that report on the levels of glucose in the body • the citric acid cycle plays important anabolic roles in the cell: Anaplerosis • organisms have multiple ways to replenish intermediates that are used in other pathways: Lipid and Nitrogen biosynthesis…....... 2 ANABOLISM II: Biosynthesis of Fatty Acids & Lipids ANABOLISM II: Biosynthesis of Fatty Acids & Lipids Lipid Biosynthesis Fatty Acid Synthesis Isoprene Biosynthesis Fat Catabolism Fatty Acid Degradation Ketone body Utilization 3 Fatty Acid Biosynthesis Catabolism Anabolism • Contrast with Sugars – Lipids have hydro-carbons not carbo-hydrates – more reduced=more energy – Long-term storage vs short-term storage – Lipids are essential for structure in ALL organisms: membrane phospholipids • Catabolism of fatty acids –produces acetyl-CoA –produces reducing power (NADH, FADH2) –takes place in the mitochondria • Anabolism of fatty acids –requires acetyl-CoA and sufficient carbohydrates –requires reducing power from NADPH –takes place in cytosol in animals, chloroplast in plants Anabolism of fat will occur only when there is excess, carbon, electrons, and DG Acetyl-CoA & carbohydrates How is this monitored in the cell? NADPH ATP Fatty Acid Biosynthesis Cytosol for animals Fatty Acid Synthesis Chloroplast for plants Both of these compartments are where there are ample Sources of NADPH: in adipocytes: pentose phosphate pathway and malic enzyme NADPH is made as malate converts to pyruvate + CO2. in hepatocytes and mammary gland: pentose phosphate pathway NADPH is made as glucose-6-phosphate converts to ribulose 6-phosphate. in plants: photosynthesis 4 Fatty Acid Biosynthesis EXAMPLE: Synthesis of Palmitate (16:0) Where do the carbons come from? Acetyl-CoA 1. 8 acetyl-CoA x 2 carbons = 16 carbons (palmityl-CoA) 2. Longer fatty acids and desaturases use palmityl-CoA 3. 1 acetyl-CoA ”primes” the enzyme a. b. other “acetyl-CoA-derived units” are ACTIVATED by carboxylation (recall gluconeogenesis) Used to make 7 malonyl-CoAs… using? ATP & CO2 7 acetyl-CoA + 7 CO2 + 7 ATP à 7 malonyl-CoA + 7 ADP + 7 Pi ….more later 2. Seven cycles of condensation, reduction, dehydration, and reduction… using 2 NADPH to reduce the b-keto group and trans-double bond acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+ à palmitate (16:0) + 7 How is the acetyl-CoA transported out CO2 + 8 CoA + 14 NADP+ + 6 H2O of mitochondria? Fatty Acid Biosynthesis •In non-photosynthetic eukaryotes…Acetyl-CoA is made in the mitochondria •But fatty acids are made in the cytosol •So acetyl-CoA is smuggled (transported) into the cytosol (This antiporter not used as much because it moves NADH into mitochondria) – cost of 2 ATPs –Converted back to acetylCoA by citrate lyase –Therefore, cost of FA synthesis is 3 ATPs per 2-C And where does the NADPH and need for carbohydrates come from? unit ….. Where is third? 5 Fatty Acid Biosynthesis • Fatty acids are built in several passes, processing one acetate unit at a time. • The acetate is coming from activated malonate in the form of malonyl-CoA. Each pass involves reduction of a carbonyl carbon to a methylene carbon. here is the third ATP! • OMSGAP • Making the Malonyl-CoA: – Reaction carboxylates acetyl-CoA – Catalyzed by acetyl-CoA carboxylase (ACC) Fatty Acid Biosynthesis Acetyl-CoA Carboxylase Reaction: –The enzyme has three subunits: • One unit has biotin covalently linked to Lys. • Another subunit is biotin carboxylase • The third subunit is a transcarboxylase • Biotin carries CO2. • In animals, all three subunits are on one polypeptide chain. –HCO3− (bicarbonate) is the soluble source of CO2. – Two-step Rxn similar to carboxylations catalyzed by pyruvate carboxylase and propionyl-CoA carboxylase • CO2 binds to biotin. - Reaction with ATP produces carboxy-phosphate. - Activated CO2 is attached to N in ring of biotin. • Enzyme undergoes conformational change to carry carbamoyl to transcarboxylase site • CO2 attaches to acetyl-CoA, which leaves active site 6 Fatty Acid Biosynthesis Fatty Acid Synthase (FAS) • Catalyzes a repeating four-step sequence that elongates the fatty acyl chain by two carbons at each step • NADPH as as the electron donor • Overall goal: attach acetate unit (2carbon) from malonyl-CoA to a growing chain and then reduce it. • Condensation with acetate – b-ketoacyl-ACP synthase (KS) • Reduction of b-carbonyl to hydroxyl – b-ketoacyl-ACP reductase (KR) • Reaction involves cycles of four enzymecatalyzed steps: • Dehydration of alcohol to alkene – b-hydroxyacyl-ACP dehydratase (DH) – condensation of the growing chain with activated acetate (malonyl-CoA) – reduction of carbonyl to hydroxyl – dehydration of alcohol to trans-alkene – reduction of alkene to alkane • Reduction of alkene to alkane –enoyl-ACP reductase (ER) • Chain transfer/charging –malonyl/acetyl-CoA ACP transferase (M/AT) • The growing chain is initially attached to the enzyme via a thioester linkage on the acyl-carrier protein (ACP). What is this “ACP”? …let’s look at the structure. Fatty Acid Biosynthesis FAS I vs. FAS II FAS I • Single polypeptide chain in vertebrates • Leads to single product: palmitate 16:0 • C-15 and C-16 are from the acetyl-CoA used to prime the Rxn • FAS I in vertebrates and fungi FAS II • Made of separate, diffusible enzymes • Makes many products (saturated, unsaturated, branched, many lengths, etc.) • Mostly in plants and bacteria FAS I What you can’t see is the ACP…... 7 Fatty Acid Biosynthesis Acyl Carrier Protein (ACP) •The E. coli ACP is a small 77-residue protein with a a covalently attached prosthetic group, 4’phosphopantetheine, at a Ser residue. – In vertebrate FAS, it’s a domain with a flexible arm to tether the growing acyl chain •Delivers acetate (in the first step) or malonate (in all the next steps) to the fatty acid synthase enzymes •Shuttles the growing chain from one active site to another during the four-step reaction Priming FAS • Two thiols must be charged with the correct acyl groups before the condensation reaction can begin. H– H- – thiol from 4-phosphopantethine in ACP – thiol from Cys b-ketoacyl-ACP synthase (KS) M A/T • The acetyl group of acetyl-CoA is transferred to ACP. 2 CoASH – catalyzed by malonyl/acetyl-CoA transferase (MAT) – ACP passes this acetate to the Cys of the KS domain of FAS 1. – ACP –SH group is recharged with malonate from malonyl-CoA again catalyzed by MAT ACP KS Synthase Fatty Acid Biosynthesis Note that malonyl-CoA and acetyl-CoA have already been attached to complex via thioester linkages to enzyme and have shed their CoA attachments. Step 1: Condensation reaction attaches two C from acetyl group (or longer fatty acyl chain) to two C from malonyl group. - Acetoacetyl-ACP release of CO2 activates malonyl group for attachment creates b-keto intermediate (acetoacetyl-ACP) Catalyzed by b-ketoacyl-ACP synthase (KS) Step 2: First Reduction: NADPH reduces the b-keto intermediate to an alcohol. – – – carbonyl at C-3 reduced to form D-b-hydroxybutyryl-ACP NADPH is 2e− donor catalyzed by b-ketoacyl-ACP reductase (KR) D-b-hydroxybutyryl-ACP Step 3: Dehydration: OH group from C-2 and H from neighboring CH2 are eliminated, creating double bond (transalkene). – – 2 OH and H removed from C-2 and C-3 of b-hydroxybutyryl-ACP trans-D -Butenoyl-ACP (Crotonyl-ACP) to form trans-D2-butenoyl-ACP catalyzed by b-hydroxyacyl-ACP dehydratase (DH) Step 4: Second Reduction: NADPH reduces double bond to yield saturated alkane. – – Butyryl-ACP NADPH is the electron donor to reduce double bond of transD2-butenoyl-ACP to form butyryl-ACP. catalyzed by enoyl-ACP reductase (ER) 8 Fatty Acid Biosynthesis Palmitate (16:0) Thioesterase H2 O H2O Fatty Acid Biosynthesis Condensation, reduction, dehydration, reduction, translocation, ACP-loading ① ❶ ❷ ❸ ② ❹ ❺ ❻ ❼ H2O H2O Palmitate (16C) Synthesis What is this like? Protein synthesis; where the peptidyl-tRNA (acyl-ACP) puts the growing peptide onto the incoming AA-tRNA (malonyl-ACP) H2O 16H+ + 16e– 4CO2 4H2O 7 acetyl-CoA + 7 CO2 + 7 ATP à 7 malonyl-CoA + 7 ADP + 7 Pi acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+ à palmitate (16:0) + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O 8 acetyl-CoA + 14 NADPH + 14 H+ + 7 ATP à palmitate (16:0) + 8 CoA + 14 NADP+ + 7 ADP + Pi + 6 H2O If you add in the transport (8x2=16): 23 ATP 9 Fatty Acid Biosynthesis 1 2 3 4 5 6 Comparison of Fatty-Acid Degradation versus Fatty-Acid Synthesis Clinical Correlations Transketolase Deficiency TPP (ketose) (aldose) (aldose) (ketose) • This is called Wernicke-Korsakoff Syndrome • It presents as a mental disorder: memory loss and partial paralysis • Often seen in alcoholics with poor nutrition • There are no mutations in the transketolase gene, which are likely embryonically lethal. So, it appears that it arises from a deficiency in thiamine used for TPP 10 Clinical Correlations 6-phosphofructo-2-kinase/Fructose-2,6-bisphosphatase (PFK2/FBPase2) in Cancer • Cancer cells learn how to avoid necrosis and apoptosis by fighting hypoxia. • The generate most of their ATP via glycolysis: called the Warburg Effect • How they increase glycolysis is due the the induction of a different isozyme of PFK2/FBPase2 that can favor the kinase when phosphorylated by AMPdependent protein kinase (AMPK). • So, in oxidative stress cancer cells experience hypoxia, which activates AMPK, and phosphorylation of PFK2/FBPase2 activates the kinase making much more Fru2,6P2 for activating glycolysis. AMPK • The body fights cancer by making tumor- suppressors • TIGAR is induced by p53, the tumor suppressor transcription factor. • It evolved from the FBPase2 domain, and possesses activity that shuts down glycolysis in cancer cells Clinical Correlations Glucose 6-phosphate Dehydrogenase Deficiency G6PDH Deficiency is from genetic alterations of the gene for this enzyme.* Ø over 150 mutations have been described Ø Causes neonatal jaundice and hemolytic anemia Ø This disorder demonstrates the interplay of genetics and environment • Can be fatal in cases of high oxidative stress. Results in Hemolytic anemia = Oxidative Stress – certain drugs, herbicides, and some foods (fava beans) • Resistance to malaria due to high oxidative stress in red blood cells– limits replication of Plasmodia • X-linked heterozygous advantage • The most common genetic disorder on Earth 500:1 Essential for red-blood cells 11 Clinical Correlations Glucose 6-phosphate Dehydrogenase Deficiency Reactive Oxygen Species (ROS) Glutathione (GSH) Fe 2+-catalyzed Fenton reaction gECG Clinical Correlations How do we get NADPH from PPP, which is made in the cytosol, into mitochondria? • Ubiquinone is naturally “leaky” and facilitates partial reduction of nonComplex III targets. Fe2+ (aconitase) – Single electron transfers result in free radicals. • One method by which the cell can correct freeradicals is the production of reduced glutathione (GSH), which fuels the glutathione shuttle Glc 6-P Dehydrogenase (G6PD) Deficiency Reactive Oxygen Species (ROS) Can Damage Biological Macromolecules Glutathione 12