Principles of Bioenergetics

Principles of bioenergetics and fuel molecule metabolism Principles of Bioenergetics (BC, GC) • Bioenergetics/thermodynamics o Free energy/Keq: • Free energy is the energy available that can be converted to do work • ∆G = ∆H - T∆S • T is temperature in Kelvin ▪ Equilibrium constant (2 ways of getting Keq) • From an equation, Keq = products/reactants = [C]c[D]d/[A]a[B]b • From thermodynamics, ∆G˚ = -RT ln (Keq) o Derivation: ∆G = 0 at equilibrium o ∆G = ∆G˚ + RT ln Q o 0 = ∆G˚ + RT ln Qat equilibrium o ∆G˚ = -RT ln Qat equilibrium • At equilibrium: o ∆G = 0 o rforward = rbackward o Q = Keq • Keq is ratio of kforward over kbackward o If Keq > 1, then position of equilibrium is to the right, more products present at equilibrium o If Keq = 1, then position of equilibrium in center, number of products roughly equal to number of reactants o If Keq < 1, then position of equilibrium is to the left, more reactants are present at equilibrium ▪ Relationship of the equilibrium constant and ∆G˚ • ∆G = ∆G˚ + RT ln Q o Set ∆G = 0 at equilibrium o Q becomes Keq at equilibrium • 0 = ∆G˚ + RT ln (Keq) • ∆G˚ = -RT ln (Keq) o Concentration ▪ Le Châtlier’s Principle: if you knock a system off its equilibrium, it will readjust itself to reachieve equilibrium ▪ A reaction at equilibrium doesn’t move forward or backward, but the application of Le Châtlier’s principle means that it will proceed forward or backward in order to restore equilibrium o Endothermic/exothermic reactions ▪ Endothermic = energy taken up by reaction in form of heat. ∆H is positive ▪ Exothermic = energy released by reaction in form of heat. ∆H is negative ▪ Enthalpy, H, and standard heats of reaction and formation • Enthalpy, H, is heat content of a reaction. • • • ∆H is change in heat content of a reaction. + means heat is taken up, - means heat is released • Standard heat of reaction, ∆Hrxn, is change in heat content for any reaction • Standard heat of formation, ∆Hf, is change in heat content of a formation reaction • Formation reaction is where a compound or molecule in its standard state is formed from its elemental components in their standard states. Standard state is where things are in their natural, lowest energy, state. e.g. oxygen is O2 (diatomic gas) and carbon is C (solid graphite) • Unit for enthalpy is energy (J), or can be expressed as energy per mol (J/mol) o Free Energy: G ▪ Free energy is energy available that can be converted to do work ▪ ∆G = ∆H - T∆S o Spontaneous reactions and ∆G˚ ▪ Spontaneous reactions occur by themselves. ▪ They have negative ∆G. ▪ Do not assume that an exothermic reaction is spontaneous, because a large, negative ∆S can cause it to become nonspontaneous ▪ Do not assume that an endothermic reaction is nonspontaneous, because a large, positive ∆S can make it spontaneous ▪ Do not assume that spontaneous reactions will occur quickly, because it depends on kinetics Phosphoryl group transfers and ATP o ATP hydrolysis ∆G << 0 ▪ Adenosine 5’-triphosphate (ATP) serves as main source of free energy in living cells. Energy stored by ATP can be liberated through direct hydrolysis or group transfer. ▪ Direct hydrolysis of ATP consists of nucleophilic attack by H2O at γ phosphate position of ATP and cleavage of the γ - β phosphoanhydride bond. ▪ ATP + H2O → ADP + Pi ▪ ∆G˚’ = -30.5 kJ/mol o ATP group transfers ▪ Group transfer reactions involve the covalent transfer of a portion of the ATP molecule to a substrate (e.g. an enzyme active site), which in turn makes subsequent metabolic reactions involving this substrate more thermodynamically favorable. Biological oxidation-reduction o Half-reactions ▪ Oxidation half reaction describes the species that loses electrons (increases in charge). e.g. Cu → Cu2+ + 2e▪ Reduction half reaction describes the species that gains electrons (decreases in charge). e.g. Ag2+ + 2e- → Ag o Soluble electron carriers ▪ Think about electron-carrier molecules a bit like a molecular shuttle. ▪ It is the job of electron carriers to harness the electrons that are lost at each step of the breakdown process. They carry the electrons lost from the oxidation process, eventually to the final electron acceptor in the body, which is oxygen. ▪ Flow of electrons in the electron transport chain that allows production of energy to produce ATP. ▪ Water-soluble carriers are free-floating in the cytosol (e.g. NADH, FADH2, cytochrome c, etc.) ▪ Lipid-soluble carriers are embedded in membrane (e.g. CoQ, FMN, etc.) o Flavoproteins ▪ ▪ Flavoproteins are proteins that contain a nucleic acid derivative of riboflavin: the Flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) They are located on the matrix face of the inner mitochondrial membrane and functions as a specific electron acceptor for primary dehydrogenases, transferring the electrons to the ubiquinone pool in the inner mitochondrial matrix Carbohydrates (BC, OC) • Description o Nomenclature and classification, common names ▪ Nomenclature: • Carbohydrate = sugars, monosaccharides, disaccharides, polysaccharides • Prefix: o Deoxy = has an -H in place of an -OH at a certain position o D/L = absolute configuration = assigned based on the chirality of the carbon atom furthest from the carbonyl group o α/β = anomeric configuration • Suffix: all sugars end in -ose ▪ Classification • Aldose = sugars with an aldehyde group • Ketose = sugars with a ketone group • Pyranose = sugars in a 6-membered ring structure = hexagonal shaped • Furanose = sugars in a 5-membered ring structure = pentagonal shaped • # ose = sugar with # carbon atoms. e.g. Hexose = sugar with 6 carbons • In order to be classified as a carbohydrate, a molecule must have: o at least a 3-carbon backbone o an aldehyde or ketone group o at least 2 hydroxyl groups ▪ Common Names • The simplest, smallest carbohydrates are glyceraldehyde and dihydroxyacetone • The 3 common monosaccharides are glucose, fructose, and galactose. Glucose is our blood sugar and the product of photosynthesis. Fructose is the sugar in fruits, and it is sweeter than glucose. Galactose is one of the monomers that make up lactose, which is the sugar in milk; it is less sweet than glucose. • The sugars that make up RNA is ribose and for DNA it is deoxyribose (more precisely 2’-deoxyribose because the difference is at the 2 carbon) • Sucrose is a disaccharide made from α-glucose and β-fructose joined at the hydroxyl groups on the anomeric carbons (making acetals). Sucrose is table sugar, the sugar we buy in stores. • Lactose is a disaccharide made from β-galactose and α/β-glucose joined by a 1-4 linkage • Starch = glucose molecules joined by α 1-4 linkage (amylose = unbranched; amylopectin = branched) • Glycogen = same as starch, but with additional α 1-6 linkages for branching o Absolute configuration ▪ The chiral carbon furthest from carbonyl group determines the absolute configuration L or D of the sugar ▪ If in the Fischer projection, the OH group on the chiral carbon furthest from the carbonyl is pointing left, then it’s L. If it’s pointing right, then it’s D. ▪ Note: L and D are enantiomers, not epimers. So, every chiral carbon center inverts. It’s just that you assign L and D based on the chiral carbon furthest from the carbonyl. o Cyclic structure and conformation of hexoses ▪ ▪ ▪ • • • Fructose forms a furanose when carbon 5 attacks the carbonyl carbon Glucose forms a pyranose when carbon 5 attacks the carbonyl carbon Convert a Fischer projection to Haworth (cyclic) form • -OH groups that point Left on Fischer become Up on Haworth • -OH groups that point Right on Fischer become Down on Haworth • -OH group on anomeric carbon (Fischer carbonyl) can be either Up (beta) or Down (alpha) • The CH2OH group on absolute configuration carbon (carbon 5) points up for D, and down for L ▪ In the planar conformation, everything is eclipsed ▪ In the chair conformation, everything is staggered ▪ All the conformations in between are partially eclipsed ▪ The Boat conformation has Flagpole interactions because axial groups attached to the head and tail of the boat clash ▪ The Twist-boat conformation lessens these Flagpole interactions in addition to reducing the number of eclipsed interactions o Epimers and anomers ▪ Epimers = different configuration in just one chiral carbon ▪ Anomers = different configuration in the chiral, anomeric carbon when the molecule is in the cyclic form ▪ Anomers are simply special types of epimers ▪ Epimers are simply special types of Diastereomers ▪ Don’t confuse with enantiomers (D/L configuration), in which everything changes configuration Hydrolysis of the glycoside linkage o Glycoside linkage = acetal linkage = linkage involving the hydroxyl group of the anomeric carbon o Glycoside linkage can also mean linkage between sugar and base in nucleotides o Examples of glycosidic linkages = starch, glycogen, nucleotide o Hydrolysis of the glycosidic bond has the same mechanism as hydrolysis of the acetal bond o Glycoside + H2O + catalyst → hydrolysis o Catalysts include: Amylase for starch and glycosylase for nucleotide Monosaccharides o Simple sugars, the most common of which is glucose. Have a formula (CH 2O)n, and typically contain three to seven carbon atoms. o Most oxygens in monosaccharides are found in hydroxyl (OH) groups, but one of them is part of a carbonyl (C=O) group. The position of the carbonyl (C=O) group can be used to categorize the sugars: ▪ If sugar has an aldehyde group, meaning that carbonyl C is last one in chain, it is known as an aldose. ▪ If carbonyl C is internal to the chain, so there are other carbons on both sides of it, it forms a ketone group and the sugar is called a ketose. o Sugars are named according to their number of carbons: some of the most common types are trioses (three carbons), pentoses (five carbons), and hexoses (six carbons) Disaccharides o Form when two monosaccharides join together via a dehydration reaction, also known as a condensation reaction or dehydration synthesis. In this process, the hydroxyl group of one monosaccharide combines with the hydrogen of another, releasing a molecule of water and forming a covalent bond known as glycosidic linkage. o Common disaccharides include lactose, maltose, and sucrose. Lactose consists of glucose and galactose and found naturally in milk. Many people can’t digest lactose as adults, resulting in • lactose intolerance. Maltose, or malt sugar, is made of two glucose molecules. Most common disaccharide is sucrose (table sugar), made of glucose and fructose. Polysaccharides o Long chain of monosaccharides linked by glyosidic bonds. The chain may be branched or unbranched and may contain different types of monosaccharides. o Common examples in living organisms include starch, glycogen, cellulose, and chitin. o Starch is the stored form of sugars in plants and made up of a mixture of two polysaccharides, amylose and amylopectin (both polymers of glucose). Plants able to synthesize glucose using light energy gathered in photosynthesis, and excess glucose, beyond the plant’s immediate energy needs, is stored as starch in different plant parts. o Cellulose is major component of plant cell walls, rigid structures that enclose the cells. Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway (BIO, BC) • Glycolysis (aerobic), substrates and products o Glycolysis = convert glucose (6 carbons) to 2 molecules of pyruvate (3 carbons) ▪ Key enzymes: Hexokinase, Phosphofructokinase, Pyruvate Kinase ▪ Location: cytosol ▪ 2 net ATP made for every glucose (2 input ATP, 4 output ATP) ▪ 2 NADH made for every glucose ▪ Occurs under both aerobic and anaerobic conditions ▪ Glycolysis inhibited by ATP o Aerobic decarboxylation (mitochondrial matrix) = convert pyruvate (3 carbons) to an acetyl group (2 carbons) ▪ Key enzyme: Pyruvate dehydrogenase ▪ 1 NADH made for every pyruvate ▪ Only occurs in the presence of oxygen ▪ Acetyl group attaches to Coenzyme A to make acetyl CoA o Feeder pathways: glycogen, starch metabolism • Fermentation (anaerobic glycolysis) o Key enzyme: Lactate dehydrogenase o 1 NAD+ made for every pyruvate o Alcohol fermentation = pyruvate reduced to ethanol o Lactic acid fermentation = pyruvate reduced to lactate o Purpose: to regenerate NAD+, which is needed for glycolysis • Gluconeogenesis (BC) o Reversal of glycolysis. Instead of using carbohydrates to produce glucose, our body converts non-carbohydrate sources (like amino acids) in our liver into glucose. o Step 1 (first irreversible step): We start with two pyruvate molecules that came from a noncarbohydrate source. Pyruvate (3 carbons) is combined with bicarbonate to create oxaloacetate (4 carbons) through pyruvate carboxylase. This reaction requires ATP. Another GTP is then used to transform oxaloacetate into phosphoenolpyruvate, a 3-carbon molecule with a phosphate group attached through phosphoenolpyruvate carboxykinase. o Step 2: Hydroxyl group is used to change the 3-carbon molecule, preparing for a phosphate group to be transferred to another carbon in the molecule. o Step 3: ATP is used to add another phosphate group to the 3-carbon molecule. o Step 4: The high energy electrons carried by NADH are used to remove one of the phosphate groups. Once NADH has lost its high energy electrons (oxidation) it’s called NAD+. o Steps 1-4 occur twice, each time with a pyruvate molecule. We need enough carbons to eventually get a 6-carbon molecule. o Step 5: The two 3-carbon molecules are attached back together, forming one 6-carbon molecule with two phosphate groups. • • o Step 6 (second irreversible step): Enzyme is used with water to create the single phosphorylated 6-carbon molecule through Fructose-1,6-bisphosphatase. The molecule is then rearranged before the final step. o Step 7 (third irreversible step): Another enzyme uses water to remove the phosphate group to produce glucose through Glucose-6-phosphatase! Pentose phosphate pathway (BC) o Key enzyme: Glucose-6-phosphate dehydrogenase o Can use any available molecules of glucose-6-phosphate, whether they are produced by glycolysis or other methods. o Takes place in the cytosol of the cell, same location as glycolysis. Two most important products are ribose-5-P sugar used to make DNA and RNA, and the NADPH molecules which help with building other molecules. o The oxidative phase: made up of 2 irreversible steps ▪ 1. G6P oxidized to form lactone. NADPH produces as a byproduct of this reaction as NADP+ is reduced as G6P is oxidized. Another reaction, catalyzed by different enzyme, uses water to form 6-phosphogluconate, the linear product. NADPH often used in reactions that build molecules and occurs in a high concentration in the cell. ▪ 2. A carbon is cleaved, and CO2 is released. Once again, the electrons released from this cleavage is used to reduce NADP+ to NADPH. This new 5-carbon molecule is called ribulose-5-phosphate. o Non-oxidative phase: reactions are reversible. Allows different molecules to enter the PPP in different areas of the non-oxidative phase and be transformed up until the first molecule of the non-oxidative phase (ribulose-5-P). Ribulose-5-P is the precursor to the sugar that makes up DNA and RNA, and is also a product of oxidative stage ▪ 3. Ribulose-5-P can be converted into two different 5-carbon molecules. One is the sugar used to make up DNA and RNA called, ribose-5-phosphate. Ribulose-5-P isn’t being divided because the carbon count is the same in the next step. ▪ 4. Rest of the cycle depends on the cell’s needs. Ribose-5-P is combined with another molecule of ribose-5-P to make one, 10-carbon atom. Excess ribose-5-P, which may not be needed for nucleotide biosynthesis, is converted into other sugars that can be used by cell for metabolism. ▪ 5. The 3-carbon molecule and 7-carbon molecule, from interconversion in step 4, interconvert again to make a new 4-carbon and 6-carbon molecule. The 4-carbon is a precursor for amino acids, while the 6-carbon molecule can be used in glycolysis. The same reversal of steps in option 4 can happen here as well. o Summary: ▪ Oxidative phase: • -1 H2O • +2 NADPH • +1 CO2 ▪ Non-oxidative phase: • Ribose-5-P for DNA/RNA building Net molecular and energetic results of respiration processes o Glycolysis: ▪ Net yield of 2 ATP per glucose molecule (4 total made but 2 used) ▪ Net yield of 2 NADH per glucose Principles of Metabolic Regulation (BC) • Regulation of metabolic pathways (BIO, BC) o Maintenance of a dynamic steady state ▪ • • The body continually degrades and synthesizes proteins in order to keep the organism functioning properly at a steady level. ▪ e.g. Hemoglobin: body continually and simultaneously degrades older hemoglobin and synthesizes new hemoglobin. ▪ This keeps metabolic enzymes fresh and new so there will be very few issues, if any, with aging and degrading enzymes in the pathway Regulation of glycolysis and gluconeogenesis o Think about Le Châtlier’s Principle: if there’s any change to something in equilibrium, the equilibrium will adjust to counter that change and return the system back to equilibrium o e.g. if we have large influx of glucose, this promotes glycolysis o e.g. if we have large influx of oxaloacetate, the equilibria will be pushed towards the opposite direction towards the production of glucose o Allosteric regulation: if we have lots of ATP in the cell, gluconeogenesis would be favored, and glycolysis would be inhibited. o These are fast-acting forms of regulation. o Slow-acting forms of regulation: often take advantage of transcriptional changes within the cell. Imagine an organism is in a long-term fasting state. It will want to up-regulate the transcription of enzymes that promote things like gluconeogenesis to dump glucose into the blood. It’s implied that the process of going from DNA to mRNA to enzymes will take much longer than simple Le Châtlier or allosteric regulation. This is more of an adaptive process that allows the organism to adapt to more long-term changes that it experiences in its environment. o Hormonal regulation: insulin and glucagon ▪ If blood glucose level rises, it stimulates the body to release the hormone insulin, and if the blood glucose levels decrease, it stimulates the body to release the hormone glucagon. ▪ Thus, insulin promotes glycolysis and glucagon promotes gluconeogenesis. Metabolism of glycogen o Glycogenolysis: degradation of stored glycogen occurs through action of glycogen phosphorylase and glycogen debranching enzyme. Rate limiting enzyme is glycogen phosphorylase, which converts the glucose 1-phosphate to glucose 6-phosphate. ▪ Glycogen phosphorylase breaks α-1,4 glycosidic bonds, releasing glucose 1-phosphate from periphery of granule. Cannot break α-1,6 bonds and thus stops when it nears outermost branch points. ▪ Enzyme is activated by glucagon in liver, so glucose can be provided for rest of the body. In skeletal muscle, it is activated by AMP and epinephrine, which signal that muscle is active and requires more glucose. It is inhibited by ATP. ▪ Debranching enzyme is a two-enzyme complex that deconstructs the branches in glycogen that have been exposed by glycogen phosphorylase. ▪ Breaks an α-1,4 bond adjacent to branch point and moves the small oligoglucose chain that is released to the exposed end of the other chain. Forms a new α-1,4 bond. Hydrolyzes the α-1,6 bond, releasing the single residue at the branch point as free glucose. This represents the only free glucose produced directly in glycogenolysis. o Glycogenesis: synthesis of glycogen granules. ▪ Glycogen synthase is the rate-limiting enzyme of glycogen synthesis and forms the α-1,4 glycosidic bond found in the linear glucose chains of the granule. It is stimulated by glucose 6-phosphate and insulin. It is inhibited by epinephrine and glucagon through a protein kinase cascade that phosphorylates and inactivates the enzyme. ▪ Branching enzyme is responsible for introducing α-1,6 linked branches into the granule as it grows. Hydrolyzes one of the α-1,4 bonds to release a block of oligoglucose (few glucose molecules bound together in a chain), which is then moved and added in a slightly different location ▪ 1. Glycogen synthase makes a linear α-1,4 linked polyglucose chain. 2. Branching enzyme hydrolyzes an α-1,4 bond. 3. Branching enzyme transfers oligoglucose unit and • • attaches it with an α-1,6 bond to create a branch. 4. Glycogen synthase extends both branches. Regulation of glycogen synthesis and breakdown o Glycogen phosphorylase in glycogenolysis activated by glucagon in liver. In skeletal muscle, it is activated by AMP and epinephrine. Inhibited by ATP. o Glycogen synthase in glycogenesis activated by glucose 6-phosphate and insulin. Inhibited by epinephrine and glucagon through a protein kinase cascade that phosphorylates and inactivates the enzyme. Analysis of metabolic control Citric Acid Cycle (BIO, BC) • Acetyl-CoA production (BC) o Glycolysis ▪ Pyruvate, the product from glycolysis, is transformed into acetyl CoA in the mitochondria. Through the enzyme, pyruvate dehydrogenase, which is a multi-enzyme complex, pyruvate loses a carbon to produce a new, 2-carbon molecule called acetylCoA. The carbon that is removed takes two oxygens from pyruvate with it and exits the body as carbon dioxide (CO2). CO2 is the waste product that you release when you exhale. o Aerobic decarboxylation (mitochondrial matrix) = convert pyruvate (3 carbons) to an acetyl group (2 carbons) ▪ 1 NADH made for every pyruvate. Only occurs in the presence of oxygen. Acetyl group attaches to Coenzyme A to make acetyl-CoA. o Fat metabolism ▪ A process called beta-oxidation breaks down the fatty-CoA, 2 carbons at a time, to make acetyl-CoA. β-oxidation produces acetyl-CoA and also FADH2 and NADH. The acetylCoA feeds into the Krebs cycle, and the FADH2 and NADH feed into the ETC. o Protein metabolism ▪ The carbon in the amino acid is converted to pyruvate or acetyl-CoA, (or other metabolic intermediates such as oxaloacetate), depending on what amino acid it is • Reactions of the cycle, substrates and products o Location: matrix of mitochondria o Acetyl-CoA and oxaloacetate are substrates for citrate synthase. Acetyl-CoA is the main substrate. Oxaloacetate can be produced by pyruvate carboxylase that converts pyruvate to oxaloacetate, however this is not as energy efficient. o Major product of this cycle is oxaloacetate, NADH, FADH2, ATP. • Regulation of the cycle o No hormonal control o Major form of regulation is through allosteric regulation. ▪ Acetyl-CoA is major substrate so if the body doesn’t have a lot of Acetyl-CoA the speed that NADH and FADH2 is produced will slow down. ▪ Citrate, under conditions of high ATP, generally shuttles lots of acetyl-CoA into cytoplasm for fatty acid synthesis. ▪ When our body breaks down amino acids and enter the TCA cycle, such as conversion to α-ketoglutarate. This occurs when body is starving so it wants to be able to produce more NADH and FADH2 to produce more ATP. ▪ The main important enzymes are citrate synthase, isocitrate dehydrogenase, and αketoglutarate dehydrogenase. ▪ NADH allosterically inhibits all three of these enzymes because it is a product of the overall TCA cycle. Another inhibitor is ATP, since this indicates that our body has produced enough energy. ▪ • Products can also inhibit, such as citrate which performs negatively feedback on citrate synthase and succinyl-CoA which negatively feedbacks onto α-ketoglutarate dehydrogenase, citrate synthase as well. ▪ First allosteric activator is ADP since this indicates need for energy. NAD+ for a similar reason. Another is calcium. Remember that muscle cells require an influx of calcium to contract. This is a way for the body, especially in skeletal muscles, to essentially couple muscle contraction with producing more ATP to meet the needs of those contracting muscles. It has been shown to activate isocitrate and α-ketoglutarate dehydrogenase. Net molecular and energetic results of respiration processes o Citric Acid or TCA Cycle ▪ Net yield of 2 ATP per glucose molecule (per 2 acetyl CoA) ▪ Net yield of 6 NADH and 2 FADH2 ▪ Net yield of 4 CO2 molecules Metabolism of Fatty Acids and Proteins (BIO, BC) • Description of fatty acids (BC) o Fatty acids are amphipathic molecules possessing a polar carboxylate-head group and nonpolar hydrocarbon tail. o Exist in body in 3 common forms: ▪ 1. Triglycerides (or triaglycerols) • 3 fatty acids esterified to glycerol • Nonpolar tails - used for energy storage • Digested by lipase • With low food intake, broken down by liver to form ketone bodies ▪ 2. Phospholipids • Fatty acids bound to glycerol, phosphate, and other groups • Amphipathic (hydrophilic + hydrophobic components) - used for cell membranes ▪ 3. Cholesterol • Fatty acids in ring form • Cholesterol is amphipathic. The ring is nonpolar and the -OH head group is polar • Used for steroid hormones, bile, membrane fluidity • Digestion, mobilization, and transport of fats o The small intestine is lined with specialized epithelial cells, which are special cells that absorb nutrients that line the inside of this tube. o Lipases are enzymes that break down fat molecules into smaller pieces for the cells to absorb. Some are secreted by the pancreas, others are found naturally along the border of the cells. o Fatty acids are nonpolar so they group together and want to bind to each other instead of dissolving into the aqueous environment. So, they form fatty droplets. Our body secretes bile from the liver once food enters the small intestine and breaks up the fat molecules into smaller pieces, increasing the surface area for which the lipase enzymes can act upon them. Specifically, they bind to the ester linkages by adding a water molecule. The end result is a free glycerol backbone which contains a hydroxyl group, which it accepts from water, forming a carboxylic acid group. o These molecules are small enough to diffuse into the intestinal cell. First step is to get the molecules into the small intestine to turn them back into triglycerides because you want to pack them in a compact unit, so we can send them to various tissues. A carrier molecule, called a lipoprotein, essentially packages all of the triglycerides along with any other hydrophobic substances that the body absorbs, such as cholesterol, into the core of a protein molecule. These proteins have polar heads, meaning they interact with the aqueous environment inside the blood stream. The specific name this particular lipoprotein that’s produced within the intestinal cell from absorbed fats, is called chylomicron. • o Recall that all of these cells are surrounded by capillaries, so they have a source of oxygen and nutrients, but also surrounded by specialized lymphatic capillaries called lacteals (a lymphatic capillary). The capillaries have very tiny fenestrations, or gaps, to allow molecules to be absorbed. They allow proteins and carbs to pass through, but not chylomicrons. Instead, they are taken up by lacteals, which have much larger pores relative to the capillary and thus allows chylomicrons to travel within them. o Where do they drain? Anything that goes into the lymphatic vessels will drain into veins in the left side, called the left thoracic duct (near our shoulder). Some lymph is also drained into the right thoracic duct. Veins enter the heart and the heart pumps it to the lungs, which reoxygenate the blood, and eventually the chylomicrons reach the arteries which drain into capillary beds, which is the site of absorption of fats by other tissues in the body. o There are enzymes in the capillary bed called lipoprotein lipase. They take the triglycerides contained in the chylomicron and break them up into individual fatty acids. This enzyme is also activated by insulin, which is present in your body in response to an influx of glucose right after a meal. o Perhaps the biggest absorber of the fatty acids in the capillary are adipose cells. They are specialized cells for storing fat and have small nuclei and a lot of room in their cytoplasm to store fat. They take up the fatty acids floating around and compact them down for storage, turn them back into triglycerides and store them as big, fatty droplets inside of their cells. o At the end of capillary bed, we have chylomicron remnants, or CR. The liver contains receptors that help reabsorb these CRs. All roads of digestion lead to the liver. The liver has a similar functionality to small intestine. It can package these fatty acids into specialized protein carrier molecules called VLDL, very low-density lipoprotein and refers to the density of protein to hydrophobic molecules inside of it. When VLDL reaches the capillary bed, it can be acted upon by lipoprotein lipase again. It also releases cholesterol to cells. o Back to the adipose cells. They have hormone receptors on their cell surfaces which can detect the levels of hormones that are circulating inside the body. Important because the major hormone that’s floating around after we’ve eaten is insulin. Hours after a meal, insulin decreases and glucagon increases. They signal adipose cells to release all of the fatty acids from the triglyceride molecules into the bloodstream. They are hydrophobic, so the body allows these free fatty acid molecules to travel in and alongside large proteins in the blood called albumin. These are also made by the liver. o Remember, most tissues (except RBCs and brain) can take up fatty acids and produce a lot of ATP from them to sustain them. o During fasting, when the body needs to maintain blood glucose levels for the brain and RBCs, they cannot use these fatty acids. The process of gluconeogenesis, which occurs largely in the liver, requires a lot of ATP. So, the liver takes a lot of these fats and break them down to produce the ATP necessary to produce gluconeogenesis. Process of converting glucose into fatty acids is also halted when levels of insulin fall. That’s because process of converting glucose to fatty acids also stimulated by insulin. Oxidation of fatty acids o Saturated fatty acids ▪ No double bonds ▪ Solid at room temperature ▪ Higher melting point, boiling point, and Van der Waals ▪ Maximum hydrogens, stack together o Unsaturated fatty acids ▪ 1 or more double bonds - bent or kinked structure ▪ Liquids at room temperature (= oils) o Location: β-oxidation occurs in the matrix of the mitochondria. Fatty esters get hydrolyzed into free fatty acids by lipases. e.g. triacylglycerol gets hydrolyzed into free fatty acids and glycerol. o With the help of ATP, the fatty acid is “activated” at the acid end by CoA (turns into a thioester) • • o A process called β-oxidation breaks down the fatty-CoA, 2 carbons at a time, to make acetyl CoA. o β-oxidation produces acetyl-CoA and also FADH2 and NADH. o Acetyl-CoA feeds into the Krebs cycle, and FADH2 and NADH feed into ETC. o On a per gram basis, fats give more energy than any other food source. Ketone bodies (BC) o Many of the tissues in our body that rely exclusively on glucose for the production of energy, are more flexible during starvation and start using a different fuel that our body switches to making several days after starvation called ketones. Ketones are effective, unlike fatty acids, because they are water soluble enough to cross the blood-brain barrier and allow us to produce ATP in times of starvation. o There is no type of product inhibition when it comes to fatty acid oxidation. So, acetyl-CoA levels can continue to rise. If fatty acid oxidation produces enough ATP in our cells, it’ll feed back onto the ETC to slow down. This leads to an increase in reduced electron carrier molecules, like NADH, which inhibits Krebs cycle. So, acetyl-CoA molecule will no longer want to enter Krebs cycle and thus allows us to move acetyl-CoA towards production of ketones, mainly inside liver. Ketones can then leave the liver, go into the bloodstream, and other tissues like the brain can take out the ketones, convert them back into acetyl-CoA which can enter the Krebs cycle and contribute to the production of ATP. Anabolism of fats (BIO) o Acetyl-CoA, the two-carbon molecule located in the mitochondrial inner matrix, is a precursor for fatty acid synthesis. There are no proteins to shuttle acetyl-CoA into the cytoplasm. Citrate shuffle is able to transport citrate into cytoplasm which is broken up back into oxaloacetate and acetyl-CoA. For oxaloacetate to go back to pyruvate, we lose a carbon which is lost as a carbon dioxide. Simultaneously, we reduce a molecule of NAD+ to NADPH. o We are going to link acetyl-CoA together to form a large fatty acid chain. Palmitic acid (16carbon fatty acid chain) happens to be the primary product of fatty acid synthesis in the body. This requires eight acetyl-CoA molecules (since they are 2 carbons each). 7 ATP molecules are required for this reaction. We need 14 NADPH molecules to reduce the carbonyl group in acetylCoA to just carbon-carbon bonds that are essentially attached to hydrogen only. We end up with 7 ADP and Pi and 14 NADP+. We also lose 6 water molecules. o First step of fatty acid synthesis is to charge up acetyl-CoA into a higher energy molecule called malonyl-CoA. This is thermodynamically unfavorable, so need to couple with ATP hydrolysis. We charge it up with a carbon dioxide molecule. o Now, we are ready to link the malonyl-CoA together to form the 16-carbon chain. The carbon dioxide plops off again to make this reaction thermodynamically favorable. We need the NADPH molecules to reduce the carbon double bonds. The addition of two NADPH molecules, loss of one water molecule, and loss of carbon dioxide group is for each subsequent addition of the two carbon subunits. o The first enzyme that carries out this activation step is acetyl-CoA carboxylase. It adds a carboxy group to the acetyl-CoA. It is the rate-limiting step of this entire fatty acid synthesis pathway. Thus, it is important to regulate this through allosteric and hormonal regulation. Citrate is an allosteric activator. Insulin activates this pathway. Long-chain fatty acids can inhibit this enzyme through product inhibition. Glucagon is a hormonal inhibitor, it signals adipose cells to release fatty acids for our cells to break down. o The enzyme that polymerizes the malonyl-CoA subunits together is called fatty acid synthase. It contains two identical subunits, each having a thiol group. Acetyl-CoA attaches to one and malonyl-CoA attaches to the other. The decarboxylation makes the carbon very nucleophilic and thus wants to attack the other carbon that is nearby, forming a carbon-carbon bond. The ultimate fate of these fatty acids is to be attached to a glycerol backbone to form a triacylglycerol molecule, that are sent off by the liver via VLDL to deliver these fats to rest of our tissues. • • Non-template synthesis: biosynthesis of lipids and polysaccharides (BIO) o Refer to above for biosynthesis of lipids o Hemiacetal formation = -OH attacks carbonyl group - produces ring form o Acetal formation = another -OH attack on same carbonyl group = produces polysaccharides if the -OH is from another monosaccharide o Reduction turns monosaccharides into polyalcohols Metabolism of proteins o Proteins are broken down into amino acids by peptidases. o The nitrogen in the amino acid is converted to urea (for desert animals, it is uric acid) o The carbon in the amino acid is converted to pyruvate or acetyl-CoA, (or other metabolic intermediates such as oxaloacetate), depending on what amino acid it is o The carbon products from amino acid metabolism can either feed into the Krebs cycle, fatty acid synthesis, or be the starting material for gluconeogenesis Oxidative Phosphorylation (BIO, BC) • Electron transport chain and oxidative phosphorylation, substrates and products, general features of the pathway o Location: the cristae (inner membrane of mitochondria) o Input NADH o Proton gradient o The electron transport chain (ETC) is essentially a series of redox reactions, where NADH gets oxidized to NAD+ and O2 gets reduced to H2O o The series of redox reactions consists of electrons passing from NADH to FMN, to Coenzyme Q, iron-sulfur complexes, and cytochromes (cytochrome b, c and aa3) before finally being used to reduce oxygen o NADH is highest in energy, while O2 is lowest in energy. When electrons are passed from NADH down a series of proteins and finally to O2, energy is released o FADH2 is lower in energy than NADH, that’s why it releases less energy when oxidized o FADH2 skips FMN and passes its electrons to Coenzyme Q o Energy released from these reactions generates a proton gradient, which drives ATP synthase to make ATP. This is called oxidative phosphorylation. o Proton gradient ▪ The energy released from passing electrons down the ETC is used to pump protons into the intermembrane space of the mitochondria ▪ H+ concentration is very high in the intermembrane space (higher than those in the matrix). Thus, this establishes an electrochemical gradient called the proton gradient. H+ wants to migrate down the proton gradient (from the IMS back into the matrix), but it can only do this by going through ATP synthase ▪ Like a water mill, ATP synthase harnesses the energy of the falling protons to convert ADP into ATP o ETC is inhibited by certain antibiotics, by cyanide, azide, and carbon monoxide • Electron transfer in mitochondria o NADH, NADPH ▪ NADH utilized in cellular respiration. It is produced in catabolic reactions and later used in ETC to obtain energy by converting NADH back to NAD+. ▪ NADPH primarily produced in the oxidative part of the PPP. NADPH used in a) anabolic synthesis to produce cholesterol, fatty acids, transmitter substances and nucleotides b) detoxifying processes as an antioxidant. ▪ NADH and NADPH are both reductive agents. o Flavoproteins ▪ • • • • Flavoproteins are proteins that contain a nucleic acid derivative of riboflavin: the Flavin adenine dinucleotide or flavin mononucleotide ▪ They are located on the matrix face of the inner mitochondrial membrane and functions as a specific electron acceptor for primary dehydrogenases, transferring the electrons to the ubiquinone pool in the inner mitochondrial matrix o Cytochromes ▪ Compounds consisting of heme bonded to a protein. ▪ They function as electron transfer agents in many metabolic pathways, especially cellular respiration. ▪ Cytochrome c transfers electrons from complex III to complex IV in ETC. It can only carry 1 e- at a time. ATP synthase, chemiosmotic coupling o Proton motive force ▪ The energy released from passing electrons down the ETC is used to pump protons into the intermembrane space of the mitochondria ▪ H+ concentration is very high in the intermembrane space (higher than those in the matrix). Thus, this establishes an electrochemical gradient called the proton gradient. H + wants to migrate down the proton gradient (from the IMS back into the matrix), but it can only do this by going through ATP synthase ▪ Like a water mill, ATP synthase harnesses the energy of the falling protons to convert ADP into ATP o Hydrogen going through ATP synthase is considered chemiosmosis. o Oxidation generates energy by the pushing of hydrogens out. Phosphorylation happens as the hydrogens experience chemiosmosis and go back in to turn the axle (F1 ATPase) and push the ADP and phosphate groups together. o Substrate phosphorylation, on the other hand, is involved in glycolysis and is where you have an enzyme directly helping to peruse the ATP without any type of chemiosmosis or proton gradient. Net molecular and energetic results of respiration processes o Electron Transport Chain ▪ Net yield of 34 ATP per glucose molecule (never achieved because leaky mitochondrial membranes result in lower yields of ATP, so usually ~32 ATPs made) ▪ 6 H2O formed when electrons unite with O2 at the end of ETC Regulation of oxidative phosphorylation o Major regulator is looking at the energy needs of the cells. If body has a lot of ATP, oxidative phosphorylation slows down. However, if we have lots of ADP, oxidative phosphorylation will be upregulated. o Although the presence of NADH is important as it is a substrate, ADP levels are more likely to alert the ETC. ATP levels are a limiting factor that alerts the ETC as compared to NAD+ levels. The body keeps NAD+ and NADH in a pretty stable ratio. Mitochondria, apoptosis, oxidative stress (BC) o Mitochondria is site of ATP production: an apparatus called the ATP synthase makes ATP from ADP by utilizing the proton gradient as the driving force. ▪ Self-replication; have own DNA and ribosomes • Mitochondria replicate independently from the cell containing the mitochondria • Mitochondria do not share the same genome with its host • Mitochondria have their own ribosomes, which are different from the host’s ribosomes in both sequence and structure • All serve to support the endosymbiosis theory ▪ Inner and outer membrane • Inner membrane surrounds the matrix • Folds of the inner membrane make up the cristae • Between the outer and inner membrane is the intermembrane space • Intermembrane space is high in protons H+ • Outer membrane separates the mitochondria from cytoplasm o Apoptosis (Programmed Cell Death) ▪ Apoptosis = death that is clean and healthy ▪ Apoptosis = activation of caspases that digest the cell from within ▪ No spilling of cell contents ▪ Afterwards, the apoptosed cell releases chemicals that attract macrophages, and gets engulfed ▪ Apoptosis can be brought upon by development or immune response (infected/cancerous cells killed by cytotoxic T cells/natural killer cells) Hormonal Regulation and Integration of Metabolism (BC) • Higher level of integration of hormone structure and function o The endocrine gland is the main structure responsible for hormonal secretion in the endocrine system. • Tissue specific metabolism o Absorptive metabolic state: series of metabolic reactions that your body does when food is in plenty. ▪ Liver: glucose is transported to the liver. It can be stored as glycogen or converted into triglycerides, storage form of fat. Glucose must be converted into glycerol, as well as fatty acids. In the liver, we don’t store triglycerides. It’s stored mostly in adipose tissues and triglycerides can’t be transported in the blood. So, they have to be converted into VLDL, which can be exported out of liver and into blood. Liver breaks down amino acids into keto acids. These α-keto acids give off ammonia when is excreted as waste, in the form of urea. If we want to store the energy that’s in the keto acids, we convert them to fatty acids and those can be converted into triglycerides, which are later stored in adipose tissues. If liver needs energy during this Absorptive State, the keto acids can be broken down into Acetyl CoA and that Acetyl CoA goes through TCA and ETC to produce ATP. ▪ Adipose tissue: glucose needs to be turned into triglycerides. The VLDL, from earlier, will be turned into fatty acids which are then combined with glycerol to form triglycerides. ▪ Muscle: glucose can be taken up and has two pathways: can convert to its storage form in glycogen or converted to pyruvate if energy is needed. Muscle also takes up amino acids from proteins and are just stored as protein in the muscle. ▪ Brain: brain consumes vast amount of energy. So, the glucose from the blood is taken up, converted to Pyruvate, and produces ATP so brain can do work. o Post absorptive state: body needs to take the stored energy and use it ▪ Liver: glycogen is going to be broken down into glucose and glucose can then be exported out of the Liver into the blood and be used for energy all over the body. Amino acids are still taken up by Liver and converted into Keto Acids. They will be used to make glucose that can be exported and used by other parts of the body, some broken down to Acetyl-CoA to produce ATP for liver energy. Triglycerides will be converted into glucose. Fatty acids can also undergo second reaction, such that they can be broken down to form Ketones. They can be used in the brain. ▪ Adipose: Triglycerides are broken down into glycerol and fatty acids can be exported into the blood and brought to the Liver to make more glucose. ▪ Muscle: protein broken down into its amino acids which are exported into the blood and, just like the glycerol and fatty acids in adipose tissue, make their way to the Liver to be converted to α-keto acids then glucose. Glycogen converted to glucose converted to • • Pyruvate to create Acetyl-CoA and produce energy for the muscles. If muscles low on oxygen, glucose can produce lactate (not as efficient and disturbs pH balance of blood). ▪ Brain: brain just always uses energy. Brain takes glucose from blood, converts to Pyruvate and goes through cellular respiration to produce ATP. Ketones from Liver also used by Brain for energy in Post Absorptive State. Hormonal regulation of fuel metabolism o Glucagon = increases blood sugar (break down glycogen, stimulate gluconeogenesis) ▪ Typically, abundant when body is fasting o Insulin = lower blood sugar (stimulates glucose uptake by cells) ▪ Typically, abundant after a meal Obesity and regulation of body mass o Insulin, leptin, and ghrelin help regulate whether the energy or food that we’ve consumed is stored or if we let it pass through and become waste. The things that we do to get rid of energy or consume energy include basal metabolic rate or amount of energy we burn at rest. o Leptin is a hormone that lives in our fat tissue, our adipose tissue, and goes to hypothalamus, part of the brain that tells us we’re not hungry o Highlights importance of having correct hormones flowing through bloodstream to talk to the brain based on the nutrients or diet you have to regulate your body mass.