Bioenergetics: How the body converts food to energy K. Dunlap

Metabolism Metabolism: Metabolism: the sum of all chemical reactions involved in maintaining the dynamic state of a cell or organism. – Pathway: – Pathway: a series of biochemical reactions. – Catabolism: – Catabolism: the biochemical pathways that are involved in generating energy by breaking down large nutrient molecules into smaller molecules with the concurrent production of energy. Anabolism: – Anabolism: the pathways by which biomolecules are synthesized.

Metabolism – Metabolism is the sum of catabolism and anabolism.

A Mitochondrion organelles in which the common catabolic pathway takes place in higher organisms; the purpose of this catabolic pathway is to convert the energy stored in food molecules into energy stored in molecules of ATP.

Common Catabolic Pathway The two parts to the common catabolic pathway: – The citric acid cycle – The citric acid cycle, also called the tricarboxylic acid (TCA) or Krebs cycle. – Electron transport chain phosphorylation oxidative phosphorylation – Electron transport chain and phosphorylation, together called oxidative phosphorylation. Four principal compounds participating in the common catabolic pathway are: – AMP, ADP, and ATP – NAD + /NADH – FAD/FADH 2 – coenzyme A; abbreviated CoA or CoA-SH

Adenosine Triphosphate ATP ATP is the most important compound involved in the transfer of phosphate groups. – ATP contains two phosphoric anhydride bonds and one phosphoric ester bond.

ATP – Hydrolysis of the terminal phosphate (anhydride) of ATP gives ADP, phosphate ion, and energy. – Hydrolysis of a phosphoric anhydride liberates more energy than hydrolysis of a phosphoric ester. – We say that ATP and ADP contain two high-energy phosphoric anhydride bonds. – ATP is a universal carrier of phosphate groups. – ATP is also a common currency for the storage and transfer of energy.

NAD + /NADH 2 Nicotinamide adenine dinucleotide (NAD + ) Nicotinamide adenine dinucleotide (NAD + ) is a biological oxidizing agent.

NAD + /NADH – NAD + is a two-electron oxidizing agent, and is reduced to NADH. – NADH is a two-electron reducing agent, and is oxidized to NAD +. – NADH is an electron and hydrogen ion transporting molecule.

FAD/FADH 2 Flavin adenine dinucleotide (FAD) Flavin adenine dinucleotide (FAD) is also a biological oxidizing agent.

FAD/FADH 2 – FAD is a two-electron oxidizing agent, and is reduced to FADH 2. – FADH 2 is a two-electron reducing agent, and is oxidized to FAD.

Coenzyme A Coenzyme A (CoA) Coenzyme A (CoA) is an acetyl-carrying group. – Like NAD + and FAD, coenzyme A contains a unit of ADP CoA-SH – CoA is often written CoA-SH to emphasize the fact that it contains a sulfhydryl group. – The vitamin part of coenzyme A is pantothenic acid. – The acetyl group of acetyl CoA is bound as a high- energy thioester.

Coenzyme A

Citric Acid Cycle – Overview: the two carbon acetyl group of acetyl CoA is fed into the cycle and two CO 2 are given off. – There are four oxidation steps in the cycle. Per turn: 3 NADH 1 FADH 2 1 GTP 2 CO 2

************************************************************  One high energy compound (GTP) is produced for each cycle.  The TCA cycle provides reduced electron carriers in the form of three NADH and one FADH 2 and ultimately energy is provided for oxidative phosphorylation. ************************************************************

******************************************* The cycle also supplies some precursors for several anabolic processes. All enzymes are in the mitochondrial matrix or inner mitochondrial membrane ***********************************************

Citric Acid Cycle Step 1: condensation of acetyl CoA with oxaloacetate: – The high-energy thioester of acetyl CoA is hydrolyzed. – This hydrolysis provides the energy to drive Step 1. – Citrate synthase, an allosteric enzyme, is inhibited by NADH, ATP, and succinyl-CoA.

Citric Acid Cycle Step 2: dehydration and rehydration, catalyzed by aconitase, gives isocitrate. – Citrate and aconitate are achiral; neither has a stereocenter. – Isocitrate is chiral; it has 2 stereocenters and 4 stereoisomers are possible. – Only one of the 4 possible stereoisomers is formed in the cycle.

Citric Acid Cycle Step 3: oxidation of isocitrate followed by decarboxylation gives  -ketoglutarate. – Isocitrate dehydrogenase is an allosteric enzyme; it is inhibited by ATP and NADH, and activated by ADP and NAD +.

Citric Acid Cycle Step 4: oxidative decarboxylation of  - ketoglutarate to succinyl-CoA. – The two carbons of the acetyl group of acetyl CoA are still present in succinyl CoA. – This multienzyme complex is inhibited by ATP, NADH, and succinyl CoA; it is activated by ADP and NAD +.

Citric Acid Cycle Step 5: formation of succinate. – The two CH 2 -COO - groups of succinate are now equivalent. – This is the first, and only, energy-yielding step of the cycle; a molecule of GTP is produced.

Citric Acid Cycle Step 6: oxidation of succinate to fumarate. Step 7: hydration of fumarate to L-malate. – Malate is chiral and can exist as a pair of enantiomers; It is produced in the cycle as a single stereoisomer.

Citric Acid Cycle Step 8: oxidation of malate. – Oxaloacetate now can react with acetyl CoA to start another round of the cycle by repeating Step 1. The overall reaction of the cycle is:

24 Reactions and enzymes of the Citric Acid Cycle

TCA Cycle in Catabolism The catabolism of proteins, carbohydrates, and fatty acids all feed into the citric acid cycle at one or more points:

Oxidative Phosphorylation Carried out by four closely related multisubunit membrane-bound complexes and two electron carriers, coenzyme Q and cytochrome c. – In a series of oxidation-reduction reactions, electrons from FADH 2 and NADH are transferred from one complex to the next until they reach O 2. – O 2 is reduced to H 2 O. – As a result of electron transport, protons are pumped across the inner membrane to the intermembrane space.

Oxidative Phosphorylation

Complex I The sequence starts with Complex I. – This large complex contains some 40 subunits, among them are a flavoprotein, several iron-sulfur (FeS) clusters, and coenzyme Q (CoQ, ubiquinone). – Complex I oxidizes NADH to NAD +. – The oxidizing agent is CoQ, which is reduced to CoQH 2. – Some of the energy released in the oxidation of NAD + is used to move 2H + from the matrix into the intermembrane space.

Complex II – Complex II oxidizes FADH 2 to FAD. – The oxidizing agent is CoQ, which is reduced to CoQH 2. – The energy released in this reaction is not sufficient to pump protons across the membrane.

Complex III – Complex III delivers electrons from CoQH 2 to cytochrome c (Cyt c). – This integral membrane complex contains 11 subunits, including cytochrome b, cytochrome c 1, and FeS clusters. – Complex III has two channels through which the two H + from each CoQH 2 oxidized are pumped from the matrix into the intermembrane space.

Complex IV – Complex IV is also known as cytochrome oxidase. – It contains 13 subunits, one of which is cytochrome a 3 – electrons flow from Cyt c (oxidized) in Complex III to Cyt a 3 in Complex IV. – From Cyt a 3 electrons are transferred to O 2. – During this redox reaction, H + are pumped from the matrix into the intermembrane space.

Coupling of Ox and Phos chemiosmotic theory: To explain how electron and H + transport produce the chemical energy of ATP, Peter Mitchell proposed the chemiosmotic theory: gradient – The energy-releasing oxidations give rise to proton pumping and a pH gradient is created across the inner mitochondrial membrane. – There is a higher concentration of H + in the intermembrane space than inside the mitochondria. proton translocating ATPase. – This proton gradient provides the driving force to propel protons back into the mitochondrion through the enzyme complex called proton translocating ATPase.

Coupling of Ox and Phos – Protons flow back into the matrix through channels in the F 0 unit of ATP synthase. – The flow of protons is accompanied by formation of ATP in the F 1 unit of ATP synthase. The functions of oxygen are: – To oxidize NADH to NAD + and FADH 2 to FAD so that these molecules can return to participate in the citric acid cycle. – Provide energy for the conversion of ADP to ATP.

Coupling of Ox and Phos The overall reactions of oxidative phosphorylation are: Oxidation of each NADH gives 3ATP. Oxidation of each FADH 2 gives 2 ATP.

The Energy Yield A portion of the energy released during electron transport is now built into ATP. – For each two-carbon acetyl unit entering the citric acid cycle, we get three NADH and one FADH 2. – For each NADH oxidized to NAD +, we get three ATP. – For each FADH 2 oxidized to FAD, we get two ATP. – Thus, the yield of ATP per two-carbon acetyl group oxidized to CO 2 is:

Other Energy Forms The chemical energy of ATP is converted by the body to several other forms of energy: Electrical energy Electrical energy – The body maintains a K + concentration gradient across cell membranes; higher inside and lower outside. – It also maintains a Na + concentration gradient across cell membranes; lower inside, higher outside. – This pumping requires energy, which is supplied by the hydrolysis of ATP to ADP. – Thus, the chemical energy of ATP is transformed into electrical energy, which operates in neurotransmission.

Other Forms of Energy Mechanical energy Mechanical energy – ATP drives the alternating association and dissociation of actin and myosin and, consequently, the contraction and relaxation of muscle tissue. Heat energy Heat energy – Hydrolysis of ATP to ADP yields 7.3 kcal/mol. – Some of this energy is released as heat to maintain body temperature.

What feeds the citric acid cycle? Glycolysis –Pyruvate –Acetyl-CoA Fatty acid oxidation Amino acid oxidation

Glycolysis is an ancient pathway that cleaves glucose (C 6 H 12 O 6 ) into two molecules of pyruvate (C 3 H 3 O 3 ). Under aerobic conditions, the pyruvate is completely oxidized by the citric acid cycle to generate CO 2, whereas, under anaerobic (lacking O 2 ) conditions it is converted to lactate. The glycolytic pathway consists of ten enzymatic steps organized into two stages. In Stage 1, two ATP are invested to “prime the pump,” and in Stage 2, four ATP are produced to give a net ATP yield of two moles of ATP per mole of glucose. Glycolysis generates metabolic intermediates for a large number of other pathways, including amino acid synthesis, pentose phosphate pathway, and triacylglycerol synthesis. Key Concepts in Glycolysis

-Glycolysis takes place entirely in the cytosol -pyruvate oxidation occurs in the mitochondrial matrix -Oxygen is not required for glycolysis in the cytosol (anaerobic) but it is necessary for aerobic respiration in the mitochondrial matrix where the O 2 serves as the terminal electron acceptor.

Glycolysis Glycolysis: Glycolysis: a series of 10 enzyme-catalyzed reactions by which glucose is oxidized to two molecules of pyruvate.

1. preparatory phase of glycolysis

2. payoff phase of glycolysis

step 1: phosphorylation of glucose Hexokinase – present in all cells (glucokinase in liver) Irreversible, rate-controlling reaction Activates glucose for subsequent reactions One ATP invested

Hexokinase binds glucose with the exclusion H 2 O from the enzyme active site and brings the phosphoryl group of ATP into close proximity with the C-6 carbon of glucose

step 2: conversion of G 6-P to F 6-P Phosphohexose Isomerase (aka: phosphoglucose isomerase) – Isomerases enzymes convert between isomers Reversible reaction Direction depends on [substrate] and [product]

step 3: phosphorylation of F 6-P to F 1,6-bisP Phosphofructokinase-1 (aka: PFK-1) Second priming reaction in preparatory phase Irreversible Rate controlling enzyme in glycolysis because the activity of PFK-1 is controlled by numerous allosteric effectors (positive and negative).

step 4: cleavage of F 1,6-bisP Aldolase (aka: fructose 1,6-bisphosphate aldolase) Rapid product removal drives the reaction The splitting of fructose-1,6-BP into the triose phosphates glyceraldehyde-3-P and dihydroxyacetone-P is the reaction that puts the lysis in glycolysis (lysis means splitting).

step 5: interconversion of triose phosphates Triose phosphate isomerase Glyceraldehyde-3-P, rather than dihydroxyacetone-P, is the substrate for reaction 6 in the glycolytic pathway, making this isomerization necessary.

step 6: oxidation of glyceraldehyde 3-phosphate to 1,3- bisphosphoglycerate Glyceraldehyde 3-phosphate dehydrogenase (a dehydrogenation) First step in the payoff phase of glycolysis Note the presence of the NAD + cofactor -The NADH formed must be re-oxidized or glycolysis will stop

step 7: phosphoryl transfer from 1,3-bisphosphoglycerate to ADP Phosphoglycerate kinase First ATP formed

step 8: conversion of 3-phosphoglycerate to 2-phosphoglycerate Phosphoglycerate mutase Mutases catalyze the transfer of functional groups from one position to another The purpose of reaction 8 is to generate a compound, 2-phosphoglycerate, that can be converted to phosphoenolpyruvate in the next reaction, in preparation for a second phosphorylation to generate ATP.

step 9: dehydration of 2-phosphoglycerate to phosphoenolpyruvate Enolase Reversible removal of water (a dehydration reaction).

step 10: transfer of the phosphoryl group from phosphoenolpyruvate to ADP Pyruvate kinase Irreversible rate controlling reaction ATP formed Unlike phosphoenolpyruvate, pyruvate is a stable compound in cells that is utilized by many other metabolic pathways.

overall balance sheet for glycolysis Glucose + __ATP + __NAD + + __ADP + 2Pi __Pyruvate + __ADP + __NADH + 2H+ + __ATP + 2H 2 O Net gain of 2 ATP per glucose in glycolysis 4-6 more ATP can be gained from the transfer of NADH to the mitochondria for oxidation there

Reactions of Pyruvate Pyruvate is most commonly metabolized in one of three ways, depending on the type of organism and the presence or absence of O 2.

What happens to pyruvate? Only 5% of total energy is released O 2 is needed as the final e - acceptor to oxidize NADH Produces the necessary NAD+

Reactions of Pyruvate A key to understanding the biochemical logic behind two of these reactions of pyruvate is to recognize that glycolysis needs a continuing supply of NAD +. – If no oxygen is present to reoxidize NADH to NAD +, then another way must be found to reoxidize it.

Pyruvate to Lactate – In vertebrates under anaerobic conditions, the most important pathway for the regeneration of NAD + is reduction of pyruvate to lactate. Pyruvate, the oxidizing agent, is reduced to lactate. – Lactate dehydrogenase (LDH

anaerobic fate: pyruvate to lactate Lactate dehydrogenase (LDH) Active skeletal muscle, erythrocytes Supplies NAD + for glyceraldehyde 3- phosphate dehydrogenase Lactate can be recycled in the liver (to glucose via the Cori cycle) Some large animals remain almost torpid until short bursts of energy are needed Extra oxygen is consumed during the long recovery period

Pyruvate to Lactate – While reduction to lactate allows glycolysis to continue, it increases the concentration of lactate and also of H + in muscle tissue – When blood lactate reaches about 0.4 mg/100 mL, muscle tissue becomes almost completely exhausted.

Pyruvate to Acetyl-CoA – Under aerobic conditions, pyruvate undergoes oxidative decarboxylation. – The carboxylate group is converted to CO 2. – The remaining two carbons are converted to the acetyl group of acetyl CoA.

Irreversible -- irreversible means acetyl-CoA cannot be converted backward to pyruvate; hence “fat cannot be converted to carbohydrate”

Energy Yield of Glycolysis