5.2 Energy Metabolism Part 2 PDF

BIOCHEMISTRY Energy Metabolism Part 2 Dr. Santos, Ricardo OXIDATIVE PHOSPHORYLATION OVERVIEW OF OXIDATIVE PHOSPHORYLATION  The process of ATP formation which is driven by energy that is ...

BIOCHEMISTRY Energy Metabolism Part 2 Dr. Santos, Ricardo OXIDATIVE PHOSPHORYLATION OVERVIEW OF OXIDATIVE PHOSPHORYLATION  The process of ATP formation which is driven by energy that is  NADH and FADH2 are produced in glycolysis, beta-oxidation of fatty released from electrons removed during substrate oxidation in the acids, TCA cycle (a.k.a Krebs cycle) and other oxidative reactions mitochondria via the respiratory chain (ETC)  The transfer of electrons from NADH to O2 occurs in stages through  The mechanism that explains oxidative phosphorylation is called large protein complexes in the inner mitochondrial membrane Chemiosmotic coupling theory  Each complex uses the energy from electron transfer to pump proteins to the intermembrane space Chemiosmotic Coupling Theory Take note that only complexes I, III, and IV uses energy from o The energy from oxidation of components in ETC is coupled to electron transfer to pump proteins to the IMS. COMPLEX II is not the translocation of protons across the inner mitochondrial included. membrane  An electrochemical potential or proton-motive force is generated (this is due to the proton pumps)  The protons re-enter the matrix through the ATP synthase (Complex V), causing ATP to be generated. This is where oxidative phosphorylation takes place In other words: The energy that is used in oxidative phosphorylation is related to the transfer of protons from the mitochondrial matrix into the intermitochondrial membrane space and it crosses the inner mitochondrial membrane o The electrochemical potential difference resulting from the symmetric distribution of the protons is used to drive the mechanism responsible for the formation of ATP Further explanation: Electrochemical potential can be broken down into: Electrical potential and Chemical potential. For electrical potential, the charge of the hydrogen ion is positive (+1). So when you transfer a positively charged proton from the matrix to the intermembrane space (IMS), Picture Above: Linking of ETC and Oxidative Phosphorylation what happens is that the matrix become electronegative (-) Without the ETC can there be oxidative phosphorylation? NO because the while the IMS is electropositive (+). This disparity in the energy that is used in oxidative phosphorylation is obtained from the energy distribution of electrical charge between the matrix and IMS released from the ETC and it is mediated by proton pump. We call that energy will produce an electrical potential difference or gradient. proton-motive force or electrochemical potential difference/gradient When you have a gradient system, you have an energized which results from the unequal distribution of protons across the inner system. mitochondrial membrane. For chemical potential, it refers to the acidity or alkalinity Can you have ETC without oxidative phosphorylation? YES because even if of the compartment of the mitochondrion. When you you remove the link between the ETC and oxidative phosphorylation transfer protons from the matrix to the IMS, the matrix will (complex V), ETC can still function. This can be exemplified when you add into be alkaline in reaction while the IMS will be highly acidic. the system called uncoupling agents (e.g. 2,4 dinitrophenol & This acidity and alkalinity of the compartment is determined pentachlorophenol). Uncoupling agents are lipid soluble, making it easy to by the concentration of the free hydrogen ion (protons). The penetrate the lipid bilayer of the inner mitochondrial membrane. Protons are more protons there is, the more acidic or lower the pH. The positively charged and charged molecules (whether + or -) are not allowed to lesser the protons, the more alkaline or higher the pH. pass through the lipid bilayer of any membrane. Uncoupling agents can bind Because of this disparity in the distribution of protons in the protons, and when they bind protons, they can carry the protons back to the compartments of the mitochondrion, that will make the matrix without passing through complex V. But the ETC will still be functional matrix alkaline in reaction, while the IMS is acidic in as long as you do not put inhibitors that will affect the flow of electrons in reaction. the ETC. As long as you have oxygen in the last complex, that will serve as the final acceptor of electrons, the ETC will still be functional. This is what electrochemical potential difference/gradient There can still be ETC even without oxidative phosphorylation. The means. This gradient system is responsible for the formation problem with this is that there is no production of ATP. If the mitochondrion of ATP. is introduced to uncoupling agents, you will not be able to produce ATP. ATP is only produced through oxidative phosphorylation. #GrindNation “Strength In Knowledge” BESHYWAP 1  BIOCHEMISTRY Energy Metabolism Part 2 Dr. Santos, Ricardo Continuation…. ENERGETICS OF MITOCHONDRIAL OXIDATION Will the uncoupling agent stop the flow of electrons in the ETC? NO, there  1 NADH or 1 FADH2 : 2 e- : ½ O2 → produce water and ATP will still be continuous flow of electrons. Release of energy is also continuous  Efficiency = P-O ratio = no. of ATPs produced in complex I, III and IV. Proton pumps are also continuously working even if O used there is uncoupling agent. In short, uncoupling agents do not disrupt continuity in ETC BUT it will stop the production of ATP P-O ratio for NADH = 2.5 P-O ratio for FADH2 = 1.5 What will happen to the energy released in the ETC, if it is not trapped (in the form of ATP)? When you synthesize ATP, the energy that is released are ELECTRON SHUTTLE SYSTEMS automatically trapped in the molecule of ATP by a process called oxidative phosphorylation. The mechanism of which is called chemiosmotic theory. So what happens to that energy is that it will just be dissipated in the form of body heat. ATP SYNTHASE (Complex V)  Functions as a rotatory motor to form ATP  Embedded in the inner mitochondrial membrane  Consists of: o Fo subcomplex – disk of C protein subunits o Gamma subunit – attached to Fo and F1 complexes o F1 subcomplex – made up of 3 alpha and 3 beta subunits  Fo – spans the membrane & serves as proton channels. Stalk is 1. Glycerol-3-phosphate shuttle system considered part of Fo. It contains the so-called oligomycin sensitivity - Simpler; uses glycerol-3-PO4+ dehydrogenase conferring enzyme (OSCP) which is the site of inhibition of proton - Major shuttle system in the brain and myocytes translocation by oligomycin - Uses FAD as mitochondrial electron carrier; this yields to 1.5  F1 – beta subunit is the site of ATP synthesis; occur by a binding moles of ATP change mechanism  3 ATPs are generated per revolution of beta subunits NAD uses DHAP and Glycerol 3-phosphate in the process so that what enters is the FADH2 2. Malate-Aspartate shuttle system - Involves transamination between oxaloacetate and glutamate - Major shuttle system in the liver and heart cells - Uses NAD as mitochondrial electron carrier; this yields 2.5 moles of ATP The form of NAD is reduced. The NADH + H is oxidized (carries Conditions Limiting the Rate of Respiration electrons and protons) but cannot enter the matrix. It will now State 1 Availability of ADP and substrate call on oxaloacetate. Oxaloacetate can carry the e- and H → State 2 Availability of substrate only reduced to malate → goes inside the matrix State 3 The capacity of the respiratory chain (ETC) itself, when all In the matrix, malate will give off the 2 e- and H to NAD substrates and components are present in saturating (regeneration of NAD) → produces oxaloacetate amounts State 4 Availability of ADP Other shuttle systems include creatine phosphate shuttle which State 5 Availability of oxygen only augments the function of creatine phosphate as an energy buffer in the heart and skeletal muscle NOTE: During exercise, cell approaches state 3 or 5 Continued next page….. #GrindNation “Strength In Knowledge” BESHYWAP 2  BIOCHEMISTRY Energy Metabolism Part 2 Dr. Santos, Ricardo INHIBITORS OF COMPLEXES Inhibitor Function Site of Action Inhibitor Function Site of Action Cyanide, Hydrogen Barbiturates sulfide, Carbon e- transport inhibitor Complex IV e- transport inhibitor (Rotenone, monoxide & Azide from Fe-S center to Complex I amobarbital or amytal, CoQ Piercidin)  No ATP production Why did ATP production stop? Answer: All of the other complexes will be oxidized; the only reduced complex  These do not totally deplete ATP production because they do not is the one that was blocked. There will be no pumping of protons once they block Complex II are oxidized → chemiosmotic theory  Electron can still be transferred through Complex II UNCOUPLERS Inhibitor Function Site of Action Inhibitor Function Site of Action 2,4 dinitrophenol, Transmembrane H+ Malonate e- transport inhibitor Complex II Uncoupling agent Pentachlorophenol carrier Uncoupling agent; Transmembrane H+ Thermogenin generate body heat carrier Oligomycin Inhibit ATP synthase OSCP of ATP synthase Uncouplers such as 2,4 dinitrophenol collapse the proton gradient by picking up protons from the IMS and release them into the matrix passing thru (diffusion) the inner mitochondrial membrane thereby bypassing ATP synthase (complex V). It equalizes the proton concentration on both sides of the membrane so that no proton-motive force is generated. Uncouplers do not disrupt the continuity of the ETC BUT they stop ATP production.  Malonic acid (malonate if ionized) is a competitive inhibitor of succinic acid (succinate dehydrogenase) Ionophores are hydrophobic molecules that dissipate osmotic gradients by  There can still be ATP production and electron transfer through inserting themselves into the membrane and forming a channel. Complex I Inhibitor Function Site of Action Antimycin A & e- transport inhibitor Complex III Dimercaprol  Medicine intake (uncoupler) → embeds itself to the inner mitochondrial membrane  Protons usually has to pass thru the ATP synthase (complex V) to go back to the matrix  But uncouplers provide another route for the protons to pass thru  Antimycin – a piscicide poison (fish poison)  Dimercaprol – used in treatment for arsenic, gold, mercury, lead and other toxic metal Continued next page…..  No ATP production #GrindNation “Strength In Knowledge” BESHYWAP 3  BIOCHEMISTRY Energy Metabolism Part 2 Dr. Santos, Ricardo CLINICAL ASPECTS 2nd Law of Thermodynamics: Law of Entropy 1. Leber’s hereditary optic neuropathy  The total entropy of a system must increase if a process is to occur o Due to mutation in mitochondrial genes that encode three spontaneously subunits of Complex I which lowers its activity  Entropy (S) is the extent of disorder or randomness of the system and o Affects CNS including the optic nerve causing sudden its total amount in nature is increasing onset blindness in early adulthood  In any spontaneous change, the amount of the free energy available 2. Fatal infantile mitochondrial myopathy & renal dysfunction decreases o Severe diminution or absence of most oxidoreductases of  The ultimate driving force of all chemical and physical processes is the ETC the tendency for the entropy of the universe to be maximized 3. Mitochondrial encephalopathy, lactic acidosis & stroke (MELAS) Example: o Inherited; due to Complex I or IV deficiency caused by a mutation in mitochondrial DNA 4. Iron deficiency anemia o Common nutritional problem o Common in menstruating and pregnant women 5. Copper deficiency in neonates Ice cube (solid), water molecules are tightly packed → water molecules will now o Leads to anemia & cardiomyopathy seek entropy or disorderliness (pool of water) → water vaper (gas), molecules 6. Ischemia/Reperfusion injury are very distant from each other → more disorderliness o Caused by occlusion in a major coronary artery during acute MI 3rd Law of Thermodynamics: State of Absolute Zero o ETC in inhibited with concomitant decrease in ATP &  Lesser known of the three major thermodynamics law activation of anaerobic glycolysis  It relates the entropy (randomness) of matter to its absolute temperature BIOENERGETICS  Refers to a state known as “absolute zero”  Biochemical thermodynamics  This is the bottom point on the Kelvin temperature scale. The Kelvin  Thermos=heat and Dynamics=power scale is absolute, meaning 0o Kelvin is mathematically the lowest  Study of energy changes accompanying biochemical reactions possible temperature in the universe which corresponds to about -  Describes the transfer and utilization of energy 273.15o C or -459.7o F  Provides the underlying principles to explain why some reactions Example: may occur while others do not As the water is cooled more, closer and closer to absolute zero, the vibration of the molecules diminishes. If the solid water reached absolute zero, all molecular LAWS OF THERMODYNAMICS motion would stop completely (water → ice). At this point, the water would have 1st Law of Thermodynamics: Law of Conservation no entropy (randomness) at all  The total energy of a system, including its surroundings, remain constant THERMODYNAMICS CONCEPTS  Energy can be converted from one form to another but CANNOT be created nor destroyed a. Enthalpy (H) – heat in a system  i.e. chemical energy → heat/electrical/radiant/mechanical energy b. Entropy (S) – randomness in a system c. Free energy (G) – energy that is free to do work Equation: ΔG = ΔH – TΔS  Expresses the relationship between the free energy change (ΔG) and the change in entropy (ΔS)  Combines the two laws of thermodynamics  ΔH – change in enthalpy (heat)  T – absolute temperature  May be expressed as ΔG = ΔE –TΔS since ΔH = ΔE  ΔE is the total change in internal energy Example: Burning log appears to create energy or destroy matter. In reality, the energy and matter are only changing place and form, they are not being created or destroyed. The wood in the log has chemical potential energy, which is released Continued next page….. when it is burned. This released energy appears in the form of heat and light. The matter of the log is changed into smoke particles, ash, and soot. The log’s total energy and mass before burning are the same as the mass and energy of the soot, ash, smoke, heat and light afterwards #GrindNation “Strength In Knowledge” BESHYWAP 4  BIOCHEMISTRY Energy Metabolism Part 2 Dr. Santos, Ricardo ΔH : Change in Enthalpy (Heat) Catabolism – breakdown or oxidation of fuel molecules; exergonic reactions  Heat released or absorbed during a reaction Anabolism – synthetic reactions that build up substances; endergonic reactions  Does not predict whether a reaction is favorable Metabolism – combined catabolic and anabolic processes  Determines whether a reaction is exothermic or endothermic  When ΔH is: COUPLED REACTION Negative reaction is EXOTHERMIC, heat will be released to the  Vital processes such as synthetic reactions, muscular contraction, surroundings nerve impulse conduction and active transport obtain energy by Positive reaction is ENDOTHERMIC, heat will be absorbed from chemical linkage or coupling to oxidative reactions the surroundings  A thermodynamically unfavorable reaction (endergonic) can be Zero reaction is ISOTHERMIC, no net exchange of heat occurs driven forward by being coupled to a thermodynamically favored with the surroundings reaction (exergonic) with an overall net ΔG is still exergonic (negative) Simply, this means that the energy that must be supplied MUST ΔS : Change in Entropy (Randomness) BE GREATER than the amount of energy that is needed by the  Measure of randomness endergonic reaction. So if there is a greater supply of energy than  That part of enthalpy which is NOT available to do work what is needed for the reaction to take place, there would be a  Does not predict whether a reaction is favorable SURPLUS energy (-ΔG) ΔG : Change in Free Energy  Free energy (G), ΔG – change in free energy  Useful energy in a system  Chemical potential  That portion of the total energy change in a system that is available for doing work  Approaches zero as reaction proceeds to equilibrium  Predicts whether a reaction is favorable or not  If ΔG is negative: Reaction proceeds spontaneously with loss of free energy Reaction is exergonic (release of energy) If ΔG is of great magnitude, the reaction goes virtually to completion Essentially irreversible  If ΔG is positive: Picture Above: Coupling of an exergonic to an endergonic reaction Reaction proceeds only if free energy can be gained A → B (releases free energy) coupled with C → D (requires free energy to Reaction is endergonic (absorption of energy) proceed) If magnitude of ΔG is great, the system is stable, with little or no A + C is the overall free energy that is available tendency for a reaction to occur B + D is the energy needed for the reaction to take place Heat is the surplus energy from the reaction  If ΔG is zero: The intersection of the exergonic (A→B) and endergonic (C→D) means that The system is at equilibrium and no net change takes place the reaction takes place simultaneously ΔGo :  ΔGo is the standard free energy change when reactants are present in concentrations of 1.0 mol/L  Standard state (represented by ‘ )– defined as having a pH of 7.0  ΔGo’ – standard free energy change at pH 7.0 Standard Free Energy Change:  Endergonic – free energy is absorbed [ΔGo’ = positive]  Exergonic – free energy is generated [ΔGo’ = negative]  Isoergonic – equilibrium, the ΔGo’ is zero With regard to spontaneity,  Endergonic reactions cannot occur spontaneously Picture Above:  Exergonic reaction can occur spontaneously This shows that exergonic (A → B) and endergonic (C → D) reaction do not take place simultaneously since they do not intersect. A → B is already taking place but C → D is not yet happening. So when you transform A → B (exergonic), you release energy. Continued next page….. #GrindNation “Strength In Knowledge” BESHYWAP 5  BIOCHEMISTRY Energy Metabolism Part 2 Dr. Santos, Ricardo Since endergonic reaction (C → D) is not taking place simultaneous with exergonic reaction (A → B), you release ALL the free energy into heat and the heat energy will go with the entropy of the system. Energy therefore is not utilized. What can be the remedy so as not to release energy as heat? The remedy is to trap that energy in a high energy compound called ATP as represented by ~(E). So the released energy from A → B will be used to phosphorylate ADP to form ATP which is a high energy compound. The ~ (wavy curve on E) is a squiggle bond which means a high energy bond. C → D, which is endergonic, will only take place when it interacts with ~(E) which is ATP Picture Above: Creatine phosphate is found in the skeletal muscles and is an energy source. The ~(P) is a high-energy phosphate bond. As you can see ~(P) is attached to creatine so it becomes creatine phosphate. When you cleave ~(P) using CK (creatine kinase), you release energy and use it to phosphorylate ADP to ATP. This exemplifies substrate level phosphorylation and as shown in the reaction, it does not involve the ETC to produce ATP. Why can you synthesize ATP from ADP using the energy provided by hydrolysis of creatine phosphate? Look at Table 11-1. In order to synthesize ATP from ADP + Pi we have to reverse the reaction ATP → ADP + Pi into ADP + Pi → ATP which can be done through substrate level phosphorylation. How much energy is needed for ADP + Pi → ATP (an endergonic reaction)? The energy needed for it to take place is +7.3 , you just have to make it positive for it to be an endergonic reaction (energy biosynthesis). A negative ΔG means that the reaction is exergonic (energy releasing). So when you hydrolyze creatine phosphate to synthesize ATP, you release -10.3 and in creating ATP from ADP you need +7.3. -10.3 - +7.3_ -3 kcal/mol -3 kcal/mol is the surplus energy or excess energy. This will be released as heat energy Picture Above: As shown in Figure 11-8, the reaction creatine phosphate → creatine is All the values that you see in the table are all negative. Why is it negative? REVERSIBLE, meaning we can turn back creatine → creatine phosphate. Whenever you hydrolyze or cleave the high-energy phosphate bonds in these The energy needed to turn back creatine to creatine phosphate is +10.3 since compounds, you release the phosphate group and energy. So all of these this is an endergonic reaction. And the reverse reaction of ATP back to ADP hydrolytic reactions are exergonic reactions, meaning they release ATP. is -7.3. Subtracting these values will yield +3 which means that the endergonic reaction will still not proceed because the free energy of the Look at the position of ATP. It is not the highest among the high-energy exergonic reaction is NOT ENOUGH. But why is it said to be reversible? It is phosphates but the lowest in the group. When you hydrolyze ATP to ADP + because of the presence of CREATINE KINASE (CK) ENZYME. Recall that Pi you release -7.3. When you hydrolyze ATP to AMP + PPi you release energy enzymes lower the energy of activation that is why the reversible reaction amounting -7.7. You see that there is PPi (pyrophosphate), part of the low- can take place. energy phosphate group, it can be further hydrolyzed and release an additional high energy bond amounting to -4.6. So if AMP + PPi is completely  The intermediate position of ATP allows it to play an important role hydrolyzed, you get -12.3. The importance of PPi (pyrophosphate) is that it in energy transfer makes a reaction irreversible  ATP allows the coupling of thermodynamically unfavorable reactions to favorable ones SUBSTRATE LEVEL PHOSPHORYLATION 1. Glucose + Pi → Glucose 6-phosphate + H2O  Most of the energy that we generate inside our body are formed (ΔGo ‘= +13.8 kJ/mol) through oxidative phosphorylation, involving the coupling of the ETC to oxidative phosphorylation where we get a P-O ratios of either 1.5 2. ATP → ADP + Pi (ΔGo’= - 30.5 kJ/mol) ATP or 2.5 ATP  In substrate level phosphorylation we only produce 1 ATP at a time Explanation:  Substrate level phosphorylation is the direct synthesis of ATP Glucose once it enters the cell is that it becomes phosphorylated through the transfer of a phosphate group from a substrate to ADP to glucose 6-phosphate and is catalyzed by glucokinase (hexokinase) in the presence of ATP. And to phosphorylate glucose → glucose 6-phosphate we need an energy of +13.8 kJ/mol. The energy released when ATP is hydrolyzed to ADP + Pi is -30.5 kJ/mol. The reaction will take place because when we compute the value we will still have a negative ΔG Many activation reactions follow this pattern #GrindNation “Strength In Knowledge” BESHYWAP 6  BIOCHEMISTRY Energy Metabolism Part 2 Dr. Santos, Ricardo ATP/ADP CYCLE  When the ß high-energy phosphate bond that is cleaved, you release this 2 Pi that are still connected by a high-energy phosphate bond. And there is a name for this 2 Pi, you call that the PPi (pyrophosphate). It is acted upon by pyrophosphatase that will cleave that high-energy phosphate bond, and that will release energy as well  So ATP consists of 2 high-energy phosphate bonds. Cleavage of each high-energy phosphate bond will give you around -7.3 and -7.7 kcal of energy/mol ADDITIONAL INFORMATION  Phosphagens act as storage forms of high-energy phosphate Examples: 1. Creatine phosphate – vertebrate skeletal muscle, heart, Spermatozoa 2. Arginine phosphate – invertebrate muscle Picture Above:  Adenyl kinase interconverts adenine nucleotides. Adenyl kinase is a Shown in red are the high-energy compounds (phosphoenolpyruvate, specialized monophosphate kinase succinyl-CoA, creatine phosphate, 1,3-Bisphosphoglucerate) and oxidative 1. ATP + AMP → 2ADP phosphorylation and these generates high-energy phosphate as represented 2. 2ADP → AMP + ATP by ~(P). These ~(P) is used to phosphorylate ADP → ATP. The ATP is then used to power all of the endergonic reactions which are boxed in blue.  Other high-energy compounds 1. Thiol esters involving coenzyme A (e.g. Acetyl CoA) ADENOSINE TRIPHOSPHATE (ATP) 2. Acyl carrier protein 3. Amino acid esters involved in protein synthesis 4. S-adenosylmethionine (SAM) 5. Uridine diphosphate glucose (UDP-Glc) 6. 5-phosphoribosyl-1-pyrophosphate (PRPP)  Other trinucleoside triphosphates also participate in the transfer of high-energy phosphates Nucleoside monophosphate kinase 1. ATP + UMP ADP + UDP Nucleoside diphosphate kinase 2. ATP + UDP ADP + UTP  Three major sources of high-energy phosphates taking part in  The sugar of ATP is ribose energy conservation or capture  The carbon atom of ribose is connected to the nitrogenous base 1) Oxidative phosphorylation – the greatest quantitative which is adenine via ß-glycosidic bond source of high-energy phosphates in aerobic organisms  Nitrogenous base + Sugar = Nucleoside 2) Glycolysis – with net formation of 2 high-energy  Nitrogenous base + Sugar + Phosphate = Nucleotide phosphates with the formation of lactate from glucose  So in forming nucleotide we add phosphate 3) Citric Acid Cycle- a.k.a. TCA cycle or Krebs cycle.  Triphosphate means to say that there are 3 phosphoric acid residues Generated directly in the cycle at the succinyl thiokinase (3 phosphate groups) step  Phosphates are named as α phosphate, ß phosphate, and γ phosphate REFERENCES  α phosphate group is connected to the 5th carbon atom of ribose via  Biochemistry Manual (2018) an ordinary phosphoester bond. It is not a high-energy phosphate  Dr. Santos Recordings bond  Harper’s Illustrated Biochemistry  2nd phosphate group which is ß phosphate is attached to the α  Shawn Juan Trans on Energy Metabolism phosphate via a high-energy phosphate bond. So you call the ß high-  PPT Notes energy phosphate bond  3rd group, γ phosphate. You have a γ high-energy phosphate bond. So when you cleave the γ high-energy phosphate bond, you remove the inorganic phosphate and you convert ATP to ADP with the concurrent release of -7.3 kcal/mol #GrindNation “Strength In Knowledge” BESHYWAP 7