Citric Acid Cycle & Mitochondria

Citric Acid Cycle Overview

• The citric acid cycle is a central metabolic pathway
• It is amphibolic, connecting the breakdown and synthesis of proteins, carbohydrates, and lipids
• Acetyl CoA serves as a common metabolic intermediate in the catabolism of carbohydrates (CH2Os), fatty acids, and amino acids
• Alternative names include the Krebs cycle (named after Sir Hans Krebs) and the tricarboxylic acid cycle (TCA cycle)

Mitochondria

• Mitochondria run the electron transport chain (ETC)
• They require a lot of oxygen
• Mitochondria are football-shaped organelles, about the size of a bacterial cell
• They posses a dual membrane structure
• The outer membrane facilitates import and export
• The inner mitochondrial membrane is highly folded, forming cristae
• The inner membrane houses the electron transport system and ATP synthase
• The space between the two membranes is the intermembrane space
• The interior is the matrix space which contains enzymes

Metabolic Pathways

• The citric acid cycle, beta-oxidation of fatty acids, and degradation of amino acids all take place here

Conversion of Pyruvate to Acetyl CoA

• Pyruvate (3 carbons) is oxidized
• This reaction produces one CO2 and one acetyl group
• The acetyl group links to coenzyme A (CoA), forming acetyl CoA which enters the citric acid cycle

Conversion of Pyruvate to Acetyl CoA Details

• This conversion is catalyzed by the pyruvate dehydrogenase complex
• It requires 5 coenzymes: CoA, TPP, lipoic acid, FAD, and NAD+
• It involves 3 enzymes: PDH, dihydrolipoyl transacetylase (TA), and dihydrolipoyl dehydrogenase (DH)
• Dihydrolipoyl transacetylase (TA), and dihydrolipoyl dehydrogenase (DH) act on lipoic acid
• The conversion is essentially irreversible
• The multienzyme complex provides advantages like, increased E-S collision and channeling of intermediates to reduce side reactions and control
• TPP** is also required

Coenzymes

• Coenzymes include oxidized NAD+/NADH(reduced) and NADP+/NADPH
• Nicotinamide adenine dinucleotide (NADPH) is a reducing agent
• NADH has 2 extra electrons and one H+ (hydride ion)
• Redox reactions are important
• Oxidation of nutrients for energy requires an electron acceptor
• The electron acceptor/oxidizing agent comes in part from niacin (vitamin B3)
• FAD/FADH2 is another coenzyme
• FADH2 has 2 extra electrons and 2 H+
• It is derived from vitamin B2
• Metabolites are oxidized with the accompanying reduction of the coenzyme
• TPP (thiamine pyrophosphate) aids in the decarboxylation of alpha-keto acids
• It is present in pyruvate dehydrogenase, pyruvate decarboxylase (ethanol fermentation), and alpha-ketoglutarate dehydrogenase complex (step 4 of TCA)

TCA Cycle

• Two more molecules of CO2 are produced for each acetyl CoA molecule that enters the cycle
• Electrons are simultaneously transferred
• The immediate electron acceptor is NAD+ (reduced to NADH), except for one step that utilizes FAD
• The only molecule entering the cycle is acetyl CoA

Reactions of the Citric Acid Cycle

• Reaction 1: A two-carbon acetyl group condenses with a four-carbon oxaloacetate to produce six-carbon citrate
  • The enzyme involved is citrate synthase with induced fit (cleft closes upon substrate binding)
  • This reaction is exergonic
  • Allosteric control by ATP, NADH, and succinylCoA
• Reaction 2: Citrate is converted to isocitrate
  • The enzyme involved is aconitase, an isomerase
  • Achiral becomes chiral
  • Hydroxyl group is moved from a tertiary to secondary alcohol, which allows oxidation
• Reaction 3: Isocitrate is converted to α-ketoglutarate
  • The enzyme involved is isocitrate dehydrogenase
  • Oxidative decarboxylation occurs in 2 steps
  • Formation of a five carbon compound, α-ketoglutarate
  • NADH is produced (hydride transfer)
  • The allosteric enzyme controls the cycle
  • Regulation by: ATP, NADH, ADP, and NAD+
• Reaction 4: α-ketoglutarate is converted to succinyl-CoA
  • The enzyme involved is α-ketoglutarate dehydrogenase complex
  • The complex consists of 3 enzymes and 5 coenzymes similar to PDH complex
  • Production of a 4-C compound through oxidative decarboxylation
  • NADH is produced
  • A high energy thioester bond is formed
• Reaction 5: Succinyl CoA is converted to succinate
  • The enzyme involved is succinyl-CoA synthetase
  • Hydrolysis of the thioester bond provides energy to phosphorylate GDP/ADP
  • Phosphate is transferred from the enzyme to GDP
  • This is the only place in the cycle where energy is directly produced
  • GTP + ADP --> GDP + ATP via nucleoside diphosphokinase
• Reaction 6: Succinate is converted to fumarate
  • The enzyme involved is succinate dehydrogenase
  • Oxidation occurs with no control
  • This happens on the inner mitochondrial membrane
  • The electron acceptor is FAD covalently bound to the enzyme
  • Only the trans product is produced
  • The rationale for these last two reactions is to regenerate OAA
• Reaction 7: Fumarate is converted to malate
  • The enzyme involved is fumarase
  • Water is added across a double bond
  • Only the L-form is produced
  • There is no control in this reaction
• Reaction 8: Malate is converted to oxaloacetate
  • The enzyme involved is malate dehydrogenase
  • The cycle is completed by the regeneration of oxaloacetate
  • NADH is produced
  • Oxidation also occurs
  • There is no control

Citric Acid Cycle Summary

• The cycle consists of eight steps, each catalyzed by a different enzyme
• Oxidation reactions occur in four steps (steps 3, 4, 6, 8)
• The oxidizing agent is NAD+ in all steps, except step 6 with FAD
• In step 5, GDP is phosphorylated to produce GTP
• Oxidation of pyruvate by the pyruvate dehydrogenase complex and the citric acid cycle generates three molecules of CO2
• The rate is controlled by the body’s cellular need for ATP
• The cycle is regulated by feedback control
• ADP builds up when energy is used at a high rate, and acts as an allosteric activator for isocitrate dehydrogenase (step 3)
• An abundance of energy leads to excess NADH, causing inhibition of isocitrate dehydrogenase

Citric Acid Cycle: Overall

• One molecule of GDP is phosphorylated, forming GTP
• One molecule of FAD is reduced, forming FADH2
• Four molecules of NAD+ are reduced, forming NADH (3 from the cycle and 1 from the complex)
• Re-oxidation of NADH and FADH2 will generate more ATPs

Role of the Citric Acid Cycle in Overall Catabolism

• Polysaccharides are hydrolyzed by specific enzymes for sugar monomers
• Lipases hydrolyze triacylglycerols, forming fatty acids and glycerol
• Proteases digest proteins, forming amino acids
• Sugars, fatty acids, and amino acids enter specific catabolic pathways as a result
• Sugars from the glycolytic pathway are converted to pyruvate (enters the citric acid cycle)
• Fatty acids are converted to acetyl-CoA and enter the cycle
• Amino acids enter the cycle through various paths

Electron Transport Chain Complexes

• Complex I:
  • A strongly exergonic reaction that releases energy to drive ADP phosphorylation to ATP
  • NADH transfers electrons to the flavin portion of flavoprotein
  • Reduced FMN is reoxidized, while the oxidized form of the iron-sulfur protein is reduced.
  • Reduction of iron-sulfur protein causes donation of electrons to CoQ to yield CoQH2
  • NADH + H+ + CoQ --> NAD+ + CoQH2
• Complex II:
  • Succinate-coenzyme Q oxidoreductase catalyzes electron transfer from succinate to CoQ
  • Succinate is oxidized to fumarate by flavin enzyme E—FAD, where flavin is covalently bound
  • The Flavin group is reoxidized as another iron–sulfur protein is reduced
  • Reduced iron–sulfur protein donates its electrons to oxidized CoQ, which is reduced
  • Succinate + CoQ --> Fumarate + CoQH2
  • This is an exergonic reaction
  • There is insufficient energy to drive ATP production
  • H+ is not pumped out of the matrix during this step
• Complex III:
  • CoQH2-cytochrome c oxidoreductase catalyzes the oxidation of reduced coenzyme Q
  • Reaction products pass electrons along to cytochrome c in multiple steps
  • H+ ions pass out on the other side of the membrane
  • CoQH2 + 2 Cyt c [Fe(III)] --> CoQ + 2 Cyt c [Fe(II)] + 2H+
    • Two cytochrome c molecules are required for every coenzyme Q molecule
  • Includes two b-type cytochromes (bH and bL), cytochrome c1, and iron–sulfur proteins
  • Is an integral part of the inner mitochondrial membrane
  • Includes series of reactions in the electron transport chain to link both two-electron and one-electron transfers
  • Involves electrons flowing via a cyclic path from CoQH2, depends on three existing forms of coenzyme Q
  • One electron is passed from CoQH2 to the iron–sulfur clusters and cytochrome c1, resulting to coenzyme Q in semiquinone form
  • Semiquinone, along with both oxidized and reduced coenzyme Q, takes part in the cyclic process where two b cytochromes are both reduced and oxidized
  • Each coenzyme Q molecule loses an electron
  • Provides a mechanism for electrons to be delivered one at a time from coenzyme Q to cytochrome c1
  • This results in proton pumping as well as sufficient energy to drive ATP production
• Complex IV:
  • Cytochrome c oxidase catalyzes the transfer of electrons from cytochrome c to O2
  • 2 Cytc [Fe(II)] + 2H+ + 1/2 O2 --> 2 Cyt c [Fe(III)] + H2O
    • This results in proton pumping
  • An integral part of the inner mitochondrial membrane.
  • Contains cytochromes a and a3 as well as two Cu2+ ions for electron transport
  • Cu2+ ions are intermediate electron acceptors between cytochromes a and a3
• All complexes have iron-sulfur clusters except for complex 4

ATP Synthase

• ATP synthase (mitochondrial ATPase) is a complex protein oligomer
• Capable of producing ATP in the mitochondria
• F0 spans the membrane
  • It consists of three kinds of polypeptide chains (a, b, and c)
• F1 projects into the matrix
  • Consists of five kinds of polypeptide chains α3β3γδε in the ratio
  • This is the site of ATP synthesis

Flow of Electrons

• Electrons are passed down four complexes
• Protons are pumped across the membrane to generate an electrochemical gradient
• ATP synthase uses this gradient to produce ATP

Lipids Overview

• Lipids have a number of major biological functions

Major Biological Functions of Lipids

• As an energy source they generate 9 kcal of energy per gram
• Can be stored as triglycerides in adipocytes (fat cells)
• Phosphoglycerides, sphingolipids, and steroids are structural components of cell membranes
• Steroid hormones serve as key intercellular messengers
• Are a source of lipid-soluble vitamins (A, E, D, K)
• Dietary fats transport lipid-soluble vitamins into cells of the small intestine
• Lipids facilitate shock absorption and promote insulation

Classification

• Includes: fatty acids, glycerides, and non-glyceride lipids

Fatty Acids Details

• Consist of long, straight-chain carboxylic acids without branching
• Chains range from 10–20 carbons in length
• Consist of an even number of carbons including the carboxyl carbon
• Saturated or unsaturated
• Typically do not have other functional groups
• Essential fatty acids cannot be synthesized by the body
• Stearic acid is a typical saturated fatty acid
• It has 18 carbons with hydrogen saturation of all carbons
• Oleic acid is a typical unsaturated fatty acid
• It has 18 carbons and at least one double bond
• Double bonds lower the melting temperature which is caused by the cis configuration that does not allow fatty acids to pack together

Glycerides

• Are lipid esters that result from the alcohol group of glycerol forming a fatty acid ester
• Esterification can occur at one, two, or all three alcohol positions producing:
  • Monoglycerides
  • Diglycerides
  • Triglycerides
• A neutral triacylglycerol (or triglyceride) serves as energy storage in adipose cells

Fats vs Oils

• Oils are a mixture of liquid triacylglycerols with a high proportion of unsaturated fatty acids
• Fats are solid mixtures of triacylglycerols with a high proportion of saturated fatty acids.
• The more double bonds a fatty acid has, the lower its melting point
• This is due to how the molecules pack together

Chemical Reactions of Triacylglycerols

• Hydrogenation: results from adding H across a double bond to produce a single bond
• Saponification: results from adding water to break a bond and produce fatty acid salts (soap)

Saponification

• Is the base-catalyzed (NaOH or KOH) hydrolysis of plant or animal fat triacylglycerols
• The hydrophobic tail binds to the dirt and moves away from the water

Soap

• The sodium salt end is ionic, hydrophilic, and dissolves in water
• The long hydrocarbon chain is nonpolar, hydrophobic, and dissolves in grease
• Tails form clusters to avoid water, which results to ionic heads making contact through the formation of micelles

Hard Water

• Causes problems when used with soaps
• It contains high concentrations of Ca2+ and Mg2+
• Cations in the water cause precipitation of fatty acid salts, interfering with the soap's emulsification forming a crusty scum

Reactions

• Reactions triglycerides undergo include: basic hydrolysis, addition etc
• Reactions are identical to carboxylic acids

Lipids: Misc

• Phospholipids have either glycerol or sphingosine backbones, two hydrophobic tails attached to a hydrophilic head group, and undergo ester linkage with phosphoric acid and an alcohol
• Glycolipids have a sphingosine backbone, contain a sugar group instead of phosphate/amino alcohol groups, may contain one sugar (cerebroside) or two sugars (ganglioside)
• Sterols consist of a common structure of 4 fused rings, they dissolve in organic solvents instead of water, and the main sterol in humans is cholesterol

Cholesterol

• Has functions as a component of cell membranes
• Starting material for all other sterols
• Some comes from food, while some is made in the liver
• It is hydrophobic except for the –OH group, and it adds rigidity to the cell membrane

Bile Acids

• Synthesized in the liver from cholesterol, and stored in the gall bladder
• Contain polar and nonpolar components
• Forms micelles where polar heads on the outside, and nonpolar ends sequestered with fats on the inside
• Ionized in the intestine, and referred to as bile salts

Steroid Hormones

• Include mineralocorticoids that regulate fluid balance between Na+ and K+ (aldosterone)
• Glucocorticoids regulate glucose metabolism (hydrocortisone)
• Sex hormones regulate secondary sex characteristics (testosterone, estradiol)

Eicosanoid Fatty Acids

• Cannot be synthesized so they are essential fatty acids
• Linoleic acid is an essential fatty acid required to generate arachidonic acid
• Arachidonic acid (20 C) is the precursor in eicosanoid production

Eicosanoid Classes

• Three structurally related classes of eicosanoid
• Prostaglandins
• Leukotrienes
• Thromboxanes

Eicosanoid Classes Functions

• Thromboxane A2 stimulates constriction of blood vessels and platelet aggregation which promotes blood clotting
• PGI2 (prostacyclin) dilates blood vessels and inhibits platelet aggregation which prevents blood clotting
• Prostaglandins mediate inflammatory responses
• PGE2 stimulates smooth muscle uterine contractions in the reproductive system
• Prostaglandins inhibit gastric secretion and increase the secretion of protective mucus in the gastrointestinal tract
• Prostaglandins dilate the renal blood vessels of the kidneys and increase water and electrolyte excretion
• Leukotrienes promote the constriction of bronchi linked to asthma
• Prostaglandins promote bronchodilation in the respiratory tract

Complex Lipids

• Are bonded to other types of molecules
• Lipoproteins transport lipids as molecular complexes that are found in the blood plasma

Lipoproteins

• Contain a neutral lipid core (cholesterol ester or triacylglycerol), surrounded by a layer of phospholipid, cholesterol, and protein

Major Classes of Lipoproteins

• Chylomicrons: very large, very low density that transport triglycerides
• VLDL: made in the liver, transports lipids to tissues
• LDL: carry cholesterol to tissues
• HDL: made in liver, scavenges excess cholesterol (good cholesterol)

Membrane Receptors

• The LDL receptor was discovered during familial hypercholesterolemia investigation
• When a cell requires cholesterol, a receptor is synthesized that will migrate to the membrane coated region
• The "captured” membrane is absorbed by endocytosis
• Failure to make the receptor is a common issue

Membrane Proteins

• Most membranes require different proteins to carry out their functions
• Integral proteins become embedded and/or extend through the membrane
• Peripheral proteins are attached by interactions with integral proteins

Lipid Metabolism

• Lipids efficiently store chemical energy

Lipid Catabolism

• Generates large quantities of energy following metabolic oxidation
• Reactions produce acetyl-CoA, NADH, and FADH2
• Fatty acids are the main source of energy (beta oxidation)
• Triacylglycerol is the main storage form in a reduced carbon form

Lipid Energy

• Energy yield per gram of fatty acid > per gram of carbohydrate
• Phosphoacylglycerols create biological membranes with multiple sites of action
• Both fatty acids and phosphoacylglycerol have fatty acids
• The bond between the fatty acid and molecule can be hydrolyzed by lipases and phospholipases

Fatty Acids Mechanism

• Activation is formed via a thioester bond to the carboxyl with CoA-SH
• Acyl-CoA synthetase forms the ester bond with ATP in the mitochondrial matrix
• Esterification occurs in the cytosol
• The rest of the reactions occurs inside the mitochandrial matrix
• Acyl-CoA can enter in the outer membrane but not the inner
• Acyl will then transfer to carnitine and can cross through the inner mito
• Transesterification reactions are catalyzed by carnitine acyltransferase

Fatty Acids Overview

• Fatty acids enter beta-oxidation by two-carbon fragments
• Activation is step 1 of beta-oxidation.
• The subsequent steps resemble the citric acid cycle
• Each pass releases acetyl-CoA
• Every cycle produces one FADH2 and NADH

Lipid Degradation

• Process where fatty acids are broken down into 2-carbon fragments which is called Beta-oxidation
• The process is activated
• Steps 2-5 involve a repetitive process of four reactions that resembles the last four of the citric acid cycle
• Each pass through the cycle causes an acetyl CoA to be released which results in a fatty acyl CoA with 2 fewer carbons
• One FADH2 and one NADH molecule are made on each beta-oxidation cycle

Lipid Oxidation

• Fatty acyl-CoA is oxidized to acetyl-CoA and reduced coenzymes
• This facilitates ATP production
• Occurs by four-step pathway
• Hydration occurs, as well as cleavage

Fatty Acid Oxidation Energy

• The # of ATPS produced is dependent on the molecules and type of acetyl-CoA produced
• The # of acetyl-CoA is dependent on the # of carbons

ATP

• Each acetyl-CoA entering the Citric Acid Cycle generates 1 ATP, 3 NADH, & 1 FADH2 = 12 ATP
• Repetition of β oxidation generates 1 NADH & 1 FADH2 = 5 ATP
• To find the amount of beta oxidations use the total number of carbon divided by 2 then subtract from that amount -1
• If there are 10 carbons, you will have half the # of acetyl coa

The Breakdown

• The overall conversion of stearic acid to produce acetyl-CoA requires 8 beta-oxidation cycles
• Each mole of stearic acid generates nine moles of acetyl CoA to enter cycle
• FADH2 and NADH produced by b-oxidation along with the citric acid cycle allows entry into the electron transport chain to generate ATP
• Overall equation of stearic oxidation obtained from the addition of b-oxidation, citric acid and oxidative phosphorylation
• Excess acetyl is converted to 3-hydroxybutyrate and acetoacetate
• To achieve balance, an excess of acetyl conversion is required

Ketone Bodies

• These are required from excess Acetyl-CoA from b-oxidation
• Glycolysis and b-oxidation create oxaloacetate convert pyruvate

Fatty Acid Biosynthesis

• Fatty acid biosynthesis (anabolism) is not the reversal of the reactions of β-oxidation
• Anabolic reactions occur in the cytosol
• Acetyl-CoA is incorporated, and may form from β-oxidation of fatty acids or by decarboxylation of pyruvate and condenses with oxaloacetate to form citrate

Protein and Amino Acid Metabolism

• Protein and amino aide metabolism is catabolized through degradation to fuel the body

Amino Acids

• Glycogen depletion causes degradation takes place in the liver in two stages
• The removal of amino involves excretion in urine, and carbon causes degradation
• The end product of protein synthesis consists of the hydrolysis of amino acids

Amino Acid Metabolism

• Located within the amino pool
• Requires all tissues and molecules to be being degraded
• Requires the cycle to continuously absorb and break apart old protein
• Biocompounds are produced
• Requires scheme for amino catabolism to remove the pool
• The pool is composed of nitrogen

Metabolism

• There various reactions that incorporate the use of the citric acid cycle
• The body reacts by excreting ammonia and can store nitrogen on the body which is toxic
• To regulate and break down synthesis transamination process occurs
• Amino group and keto group use an a-keto
• Transaminase enables transport with keto and amino molecules
• In TCA a group of amino will act to regulate
• Group amino to convert to a keto and glutamate
• Concentration varies through the process that enables regulation
• Conversion of amino acid is oxidative and removes ammonium

The Urea Cycle

• Ammonia toxicity can be avoided by amino group carriers (glutamate)
• Glutamate is useful for amino groups and synth of new aminos and recycles a-glutarate
• Urea eliminates ammonia toxicity