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BIOENERTICS

metabolism - sum total of all chemical reactions in living cells catabolic reactions - degrade macromolecules and other molecules to release energy anabolic reactions - used to synthesize macromolecules for cell growth, repair, and reproduction Can divide metabolism into 4 groups: carbohydrates, lipids, amino acids, nucleotides. within each group are a set of pathways pathways can take different forms: 1) linear - product of one reaction is substrate for another e.g. glycolysis 2) cyclic - regeneration of intermediates e.g. Krebs cycle 3) spiral - same set of enzymes is used repeatedly e.g. fatty acid synthesis, oxidation each pathway may have branch points for metabolites to enter or leave. Why have metabolic reactions with so many steps? 1) energy input and output can be controlled energy transfer occurs in discrete steps as it it transferred to acceptors a little at a time 2) enzymes can catalyze only a single step of a pathway 3) provides opportunities to establish control points, which are essential for cell function Methods of Metabolic Pathway Regulation 1) feedback inhibition product of pathway controls its own rate of synthesis occurs in the first committed step E1 E2 E3 A -----> B -----> C -----> D advantage is obvious --> prevention of intermediate accumulation 2) feed forward activation (positive feedback) metabolite produced early in pathway activates an enzyme later in pathway also prevents accumulation of intermediates E1 E2 E3 E4 A -----> B -----> C -----> D -----> E 3) allosteric activators and inhibitors 4) covalent modification addition of phosphoryl groups via protein kinases removal of phosphoryl groups via phosphatases Major Catabolic Pathways begins with extracellular digestion of polymers (exogenous) amylase in mouth and intestine work on starch protein digestion starts in stomach and finished via pancreatic proteases and intestinal peptidases lipid digestion - triacylglycerols hydrolyzed to fatty acids by phospholipases absorption occurs in intestine ---> blood ---> body can also have endogenous sources, such as glycogen and triacylglycerols catabolism yields 3 possible compounds: 1) acetyl CoA 2) nucleoside triphosphates 3) reduced coenzymes starts with glycolysis (glucose catabolism), citric acid cycle, polysaccharide mobilization, oxidative phosphorylation nucleotides are metabolized for excretion, not energy production Thermodynamics and Metabolism used to understand equilibrium and flux (flow of material through a metabolic pathway) in metabolism metabolic pathways are not at equilibrium, but at steady state (e.g. leaky bucket) free energy change ( is a measure of energy available to proceed in a chemical reaction G) G = Gproducts - Greactants at equilibrium, G = 0, no free energy available would like G to be as small as possible (i.e. negative) Free energy change of a chemical reaction is expressed in terms of changes in heat content (enthalpy) and randomness (entropy) = - T G H S H = change in enthalpy T = temperature in o Kelvin S = change in entropy when G = -, reaction is spontaneous, and no energy input is needed when G = +, must supply energy from outside

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when G = 0, reaction is at equilibrium Go = standard free energy change of a biochemical reaction at standard conditions (pH 7.0; 25oC; 1M concentration of solute)

Go of a reaction is related to Keq (equilibrium constant of a reaction) A + B ---> C + D Grxn = (GC + GD) -(GA -GB) Keq = [C][D] [A][B] Go = -2.303 RTlog Keq or Go = -RT ln Keq
-1 -1 R = gas constant 8.315 JK mol

under ideal conditions (standard conditions): if Keq > 1, Go is negative and reaction will proceed to equilibrium if Keq = 1, Go =0 and reaction is at equilibrium if Keq <1, Go is positive G and Go are related by the following equation: G = Go + RT ln Q Q = [C][D] [A][B] R= 8.315JK-1mol-1 T = 298oK (25oC) free energy change is a measure of how far from equilibrium the system is poised G, not Go determines spontaneity of a reaction and its direction means that some reactions have a -G even if under standard conditions they have a +Go. happens if Q is small or [A][B] >>[C][D] Still find reactions that have a Go that is positive and still part of a metabolic pathway How can these reactions with Go be made to go forward? 1) have other than standard concentrations 2) thermodynamic coupling Go can be summed for a series of reactions e.g. want A -----> C A -----> B + C Go = +5 kcal/mol B -----> D Go = -8 kcal/mol A -----> D + C Go = -3 kcal/mol To make C from A is thermodynamically unfavorable, but if coupled to B, then becomes favorable. 3) couple reaction to ATP hydrolysis very common ATP -----> ADP + Pi Go= -30 kJmol-1 ATP -----> AMP + PPi Go= -32kJmol-1 o AMP ----> adenosine + Pi G = -14 kJmol-1 also works with other nucleoside triphosphates, such as UTP, GTP, and GTP ADP and AMP are often allosteric modulators of some catabolic reactions ATP not effective in the role of allosteric modulator because its concentration is kept relatively constant in the cell cells typically maintain [ATP] of 2-10 mM, [ADP] <1 mM, and [AMP] <<1 mM the metabolic role of ATP to above problem: A -----> B + C Go = +5 kcal/mol ATP -----> ADP + Pi Go = -7.3 kcal/mol A + ATP------> B + C + ADP +Pi Go = -2.3 kcal/mol There are several types of group transfer reactions that involve ATP: 1) phosphoryl group transfer some metabolites have high phosphoryl group transfer potential (ability to transfer phosphoryl groups) - e.g. phosphoenolpyruvate (Go = -62 kJmol-1) transfer of phosphoryl group to ADP to form pyruvate; reaction is metabolically irreversible (Q is far from Keq) - e.g. phosphagens, such as phosphocreatine and phosphoarginine - found in animal muscle cells - phosphocreatine acts as storage of phosphoryl group by the following reaction: creatine kinase phosphocreatine + ADP -------------> creatine + ATP

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- phosphoarginine used in molluscs and arthropods 2) nucleotidyl-group transfer e.g. synthesis of acetyl CoA - AMP is transferred to nucleophilic carboxylate group of acetate --> acetyl group is transferred to sulfur atom of CoA acetyl CoA synthetase ATP + acetate + CoA -----------> AMP + acetyl CoA 3) thioesters usually make ATP equivalents succinyl CoA + GDP + Pi -----> succinate + GTP + HS-CoA

Glycolysis
Purpose: catabolism of glucose to provide ATPs and NADH molecules Also provides building blocks for anabolic pathways. Sequence of 10 enzyme-catalyzed reactions: glucose pyruvate 2 ATPs and 2 NADH produced All enzymes (and reactions) are cytosolic. Net reaction: glucose + 2ADP + 2NAD+ +2Pi 2 pyruvate + 2ATP + 2NADH +2H+ +2H2O Can catabolize sugars other than glucose: e.g. fructose ----> 2 glyceraldehyde 3-phosphate e.g. lactose --> glucose + galactose galactose --> glucose 1-phosphate --> glucose 6-phosphate e.g. mannose ---> mannose 6-phosphate --> fructose 6-phosphate Ten Steps of Glycolysis

1) glucose --> glucose 6-phosphate by hexokinase G = -8.0 kcal/mole Hexokinase also works on mannose and fructose at increased [ ]. Serves to trap glucose in the cell --> a phosphorylated molecule cannot leave 2) glucose 6-phosphate --> fructose 6-phosphate by glucose 6-phosphate isomerase Example of aldose--> ketose isomerization. Enzyme is very stereospecific. Reaction is near equilibrium in cell --> not a control point in glycolysis 3) fructose 6-phosphate --> fructose 1,6-bisphosphate by phosphofructokinase-1 (PFK-1) Reaction has G = -5.3 kcal/mole and is metabolically irreversible. Represents the first committed step in glycolysis. 4) fructose 1,6-bisphosphate --> dihydroxyacetone phosphate + glyceraldehyde 3-phosphate by fructose 1,6 bisphosphate aldolase. 5) DHAP --> glyceraldehyde 3-phosphate by triose phosphate isomerase Also catalyzes aldose--> ketose conversion. Rate is diffusion controlled (substrate is converted to product as fast as substrate is encountered). 6) glyceraldehyde 3-phosphate --> 1,3-bisphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase One molecule of NAD+ is reduced to NADH --> respiratory chain 7) 1,3 bisphosphoglycerate --> 3-phosphoglycerate Phosphoryl group transfer to ADP to form ATP. Because phosphate group comes from a substrate molecule, called substrate level phosphorylation First ATP-generating step of glycolysis. 8) 3-phosphoglycerate --> 2-phosphoglycerate by phosphoglycerate mutase Mutases are enzymes that transfer phosphoryl groups from one part of a substrate molecule to another. 9) 2-phosphoglycerate --> phosphoenolpyruvate (PEP) by enolase (forms double bond) 10) PEP --> pyruvate Second time for substrate level phosphorylation. Reaction is metabolically irreversible.

FATE OF PYRUVATE
Under anaerobic conditions, cells must be able to regenerate NAD+ or glycolysis will stop. Usually regenerated by oxidative phosphorylation, but that requires O2. There are 2 anaerobic pathways that use NADH and regenerate NAD+. 1) alcoholic fermentation Conversion of pyruvate to ethanol H+ CO2 NADH NAD+ pyruvate acetaldehyde ethanol pyruvate alcohol decarboxylase dehydrogenase glucose +2Pi + 2ADP + 2H+ ---> 2 ethanol + 2CO2 + 2ATP + 2H2O 2) lactate fermentation pyruvate NADH + H+ NAD+ ------------------------> lactate dehydrogenase lactate

glucose +2Pi + 2ADP ---> 2 lactate + 2ATP + 2H20 Lactate causes muscles to ache.Also produced by bacterial fermentation of lactose. 3) entry into citric acid cycle REGULATION OF GLYCOLYSIS First step possible is glucose transport into cells via glucose transporters. There are three enzymes that can be regulated: 1) hexokinase Catalyzes the first irreversible reaction. Inhibited by glucose 6-phosphate. Not true controlling step because not a committed step.

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Glucose 6-phosphate can be used elsewhere (i.e. pentose phosphate pathway glycogen synthesis). 2) phosphofructokinase-1 ATP is an allosteric inhibitor because it increases the Km of PFK-1 for fructose 6AMP is an allosteric activator; same for ADP When [ATP] is low, [AMP] is high --> low [ATP]/[AMP] levels stimulate PFK-1. Citrate is an allosteric inhibitor of PFK-1 (ample substrate entering citric acid pHi also regulates PFK-1 ( inhibition is due to excess H+ due to lactic acid accumulation --> no O2 present to continue). Fructose 2,6-bisphosphate is an allosteric activator. ATP ADP fructose 6-phosphate fructose 2,6-bisphosphate PFK-2 activity F 2,6-BPase activity (2 active sites on enzyme) Substrate is present in all cells but procaryotes. PFK-2 activity stimulated by Pi and inhibited by citrate. PFK-2 activity linked to action of glucagon due to adenylate cyclase activation --> phosphorylation of serine residue of PFK-2 --> inactivates kinase activity but activates phosphatase activity -> [fructose 2,6 bisphosphate] decreases as it is converted to fructose 6-phosphate , PFK-1 falls --> glycolysis decreases Glucagon is made by pancreas and is secreted when blood sugars levels fall --> mobilizes glycogen breakdown. 3) pyruvate kinase Regulated by allosteric modulation and covalent modification. Allosterically activated by fructose 1,6-bisphosphate. Allosterically inhibited by [ATP]. Protein kinase A phosphorylates pyruvate kinase --> less active. and phosphate.

cycle).

Krebs Cycle

Krebs Cycle The Krebs cycle is also known as the citric acid cycle. Citrate is a tricarboxylic acid, and the Krebs cycle is also known as the tricarboxylic acid (or TCA) cycle. It is 2nd part of aerobic respiration. It occurs in mitochondria. Pyruvic acid (3-C compound) produced in glycolysis in cytosol is converted into Acetyl Co-A by release of one CO2 molecule and formation of one molecule of NADH. The enzyme is Pyruvate dehydrogenase. Acetyl Co-A enters into mitochondria for the first reaction of Krebs cycle. Step 1: Condensation In step 1 of the Krebs cycle, the two-carbon compound, acetyl-S-CoA, participates in a condensation reaction with the four-carbon compound, oxaloacetate, to produce citrate. Citric acid synthetase Acetyl Co-A + OAA Citric Acid Step 2. Isomerization of Citrate It involves moving the hydroxyl group in the citrate molecule by a sequential dehydration and hydration reaction, to form the D-Isocitrate isomer with cis-Aconitase as the intermediate. A single enzyme, Aconitase, performs this two-step process. Citrate Cis-aconitase Isocitrate Step 3: Generation of CO2 by an NAD+ linked enzyme It is the first oxidative decarboxylation step of Krebs Cycle. The reaction is catalyzed by the enzyme Isocitrate dehydrogenase. Isocitrate is converted to a-Ketoglutaric acid. The reaction involves dehydrogenation to Oxalosuccinate, an unstable intermediate which spontaneously decarboxylates to give a-Ketoglutarate . In addition to decarboxylation, this step produces a reduced nicotinamide adenine dinucleotide (NADH) cofactor. Isocitrate Oxalosuccinate a-Ketoglutarate Step 4: A Second Oxidative Decarboxylation Step This step is performed by a multi-enzyme complex, the a-Ketoglutarate Dehydrogenation Complex. a-Ketoglutarate Succinyl Co-A Summary of reactions till Step 4 Two carbons have been added to Oxaloacetate by the action of Citrate Synthase (and Acetyl-CoA). Two carbons have been lost as CO2 by oxidative decarboxylation steps. Two oxidized NAD+ cofactors have been reduced to NADH . In the remaining steps of the Krebs cycle, the Succinyl-CoA is converted back into the original substrate for the cycle: Oxaloacetate. Step 5: Substrate-Level Phosphorylation Succinyl-CoA is a high potential energy molecule. The energy stored in this molecule is used to form a high energy phosphate bond in a Guanine nucleotide diphosphate (GDP) molecule. Succinyl Co-A synthetase Succinyl Co-A + iP + GDP Succinate 6

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Most of the GTP formed is used in the formation of ATP, by the action of Nucleoside Diphosphokinase. In plants and bacteria ATP is formed in the Succinyl-CoA Synthase catalyzed reaction by phosphorylation of ADP directly. In animals, GDP is the substrate in the reaction with formation of GTP (which is then used to form ATP by Nucleoside Diphosphokinase). Step 6: Flavin-Dependent Dehydrogenation The Succinate produced by Succinyl CoA-Synthetase in the prior reaction needs to be converted to Oxaloacetate to complete the Krebs cycle. Both Succinate and Oxaloacetate are 4-carbon compounds. The first step in the conversion is the dehydrogenation of Succinate to yield Fumarate. Succinate dehydrogenase Succinate Fumarate In this reaction a C-C bond is being oxidized to produce a C=C bond. This oxidation is energetically more costly than oxidizing a C-O bond. The redox coenzyme for this reaction is FAD, and not NAD+ (FAD is a more powerful oxidizing agent compared to NAD+). FAD is covalently bound to the Succinate Dehydrogenase molecule. Reactions at molecular level Step 7: Hydration of a Carbon-Carbon Double Bond Fumarate undergoes a stereo-specific hydration of the C=C double bond, catalyzed by Fumarate Hydratase (also known as Fumarase), to produce L-Malate. Fumarase is the enzyme. Its hydrate Fumarate, Step 8: A Dehydrogenation Reaction that Regenerates Oxaloacetate L-Malate (Malate) is dehydrogenated to produce Oxaloacetate by the enzyme Malate Dehydrogenase . This is a highly endergonic reaction, and so, the equilibrium strongly favors the reactants over the products. The step where acetyl Co-A condenses with OAA is a highly exergonic reaction catalyzed by Citrate Synthase. It keeps the level of Oxaloacetate low in the cell (mitochondrion) allowing the above reaction to proceed. Summary of Krebs Cycle in terms of ATP (energy gain) Total NADH in one Krebs cycle = 4 molecules From one glucose molecule = 8 NADH molecules. 8 NADH molecules would yield 8 x 3 = 24 ATP molecules in electron transport system. Total FADH2 in one Krebs cycle = 1 molecule From one glucose molecule = 2 FADH2 molecules. 2 FADH2 molecules would yield 2 x 2 = 4 ATP in E.T.S. ATP production at substrate level is 1 x 2 = 2 molecules from one glucose molecule. Total ATP generation from one glucose molecule in Krebs cycle source = 24 + 4 + 2 = 30 molecules.

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