MB - Lecture 5 - PDH + Citric · MetabolicBiochemistry Lecture5* * Lecture 5:The PDH Complex Part 1: Pyruvate Dehydrogenase ! Content:! 4 The!pyruvate!dehydrogenase!reaction!mechanism!!

Metabolic Biochemistry Lecture 5

Lecture 5:The PDH Complex Part 1: Pyruvate Dehydrogenase Content:

-‐ The pyruvate dehydrogenase reaction mechanism -‐ Roles of vitamin derivatives in the pyruvate dehydrogenase complex reaction

Pyruvate Dehydrogenase Complex that is a multi-‐enzyme that contains 3 types of different enzymes, E1, E2 and E3.

-‐ Responsible in generating acetyl co-‐A from pyruvate, this acetyl co-‐A enters the citric acid cycle -‐ Aerobic processes that occur in the mitochondria are pyruvate dehydrogenase, citric acid cycle and oxidation

phosphorylation Overview of oxidative respiration:

1) Pyruvate dehydrogenase: occurs in mitochondria 2) Citric acid cycle: matrix 3) Oxidative phosphorylation: inner and outer membrane

Pyruvate:

-‐ Made from glycolysis needs to be transported into the mitochondria for the next phase -‐ Pyruvate and H+ ions go through pores in the outer membrane but cannot get into inner membrane -‐ Symporter: channel in cell membrane: allows both pyruvate and H+ into the inner membrane into the matrix

as both as important in process ATP and Muscles:

-‐ Energy currency of cells -‐ Power enzyme reactions via ‘coupling’ -‐ Non-‐enzymatic reactions: muscle contraction -‐ Main difference in slow oxidative and fast oxidative is the use e.g. slow for long distance and fast for quick

movements -‐ Based on where they get ATP from -‐ 3 types of muscle fibres:

Muscle types

-‐ Difference in colour due to blood supply and myoglobin levels (stores oxygen) -‐ Slow oxidative

o Need high oxygen supply (dark red because high amount of myoglobin) o For long distance e.g. marathon

-‐ Fast glycolytic o Low mitochondria and blood supply (don’t need stored hemoglobin) o Short powerful bursts

-‐ Fast oxidative glycolytic


o Mixed o Short powerful bursts

Pyruvate entry Pyruvate à Acetyl CoA – enzyme that does this is pyruvate dehydrogenase

-‐ This releases carbon dioxide and 2 electrons -‐ Acetyl CoA is a common entry point into the citric acid cycle for protein and fats for production of energy via

aerobic respiration -‐ Only carbs can go through glycolysis to create pyruvate that can then enter pyruvate dehydrogenase

Pyruvate catabolism:

-‐ Overall reaction Pyruvate reacted with Coenzyme A via athyl – NAD à reduced to NADH (this is later used in

used in oxidative phosphorylation) -‐ One of the three carbons in pyruvate (yellow group) is released as carbon dioxide leaving 2 carbon

compound bound to coenzyme a to create Acetyl CoA -‐ Dehydrogenation (hydrogen removal) and decarboxylation (CO2 removal) of pyruvate

o Generate NADH (reduced electron carrier) -‐ Highly exergonic, Delta G of -‐33, essentially irreversible in physiological conditions – this is why fatty acids

cannot be used to produce glucose via gluconeogenesis Pyruvate Dehydrogenase complex (PDH) Co-‐enzyme: organic non-‐protein that interacts with a protein to make an enzyme PDH – interacts with 5 co-‐enzymes:

-‐ TPP: Thymine pyro phosphate – comes from thiamine or vitamin B1, in fortified bread and grain, wheat germs and pork

-‐ Lipoamide: comes from lipoic acid from green leafy vegetables, red meat and beer -‐ Coenzyme A: comes as pantothenic acid – vitamin B5; in broccoli, eggs, mushrooms and poultry -‐ FAD – riboflavin, vitamin B2 – cereals, nuts, eggs, milk, red meat, green vegies -‐ NAD+: Vitamin B3 – dairy, poultry, fish, nuts, eggs


Redox reactions – Revision: Oxidized:

-‐ Lost hydrogen -‐ Lost electron -‐ Given to reduced electron carriers (e.g. NADH)

Reduced -‐ Gained Hydrogen -‐ Gained electron -‐ Often paired with hydride ion (H, 1 hydrogen, 2 electrons) -‐ Therefore molecule is negatively charged.

TPP

-‐ Thiamine pyrophosphate o Sourced from thiamine (Vit. B1) o Acidic carbon interacts with middle carbon of

pyruvate o TPP is required for carbohydrate

metabolism – brain cannot catabolize fatty acids for energy

o Therefore brain must have a function PDH complex to turn pyruvate from glucose into acetyl CoA to enter the citric acid cycle

o Thiamine deficit leads to Beri Beri – which has symptoms of muscle weakness, paralysis or heart failure

o Causes: diet lacking thiamine (white rice, alcoholism), genetics o Treatment through foods rich in thiamine o TPP is a cofactor of the first unit in PDH – called E1

Lipoamide

-‐ Second Coenzyme, interacts with E2 of PDH -‐ Dithiol group (SS group) gets oxidized to SS or

reduced to 2 lots of SH -‐ During redox reactions, one of the thial groups

gets acetylated and this bounds to a 2 carbon compound

-‐ Permanently bound to dihydrolipoyl transacetylase (E2 of PDH)

-‐ The carboxyl group of lipolic acid binds through to the amino group of the side chain of the lysine residue (NH) in E2 forming lipoamide

Coenzyme A

-‐ Not bound to enzymes in PDH group -‐ Carries acetyl groups -‐ Binds acetate to make acetyl-‐coA -‐ Has 3 components – modified ADP, a

pantothenic acid (Vit B5) and a Beta-‐Mercapto-‐thylamine

-‐ The thiol groups in the beta Mercapto-‐thylamine section binds with acetate to create Acetyl coA


FAD

-‐ Flavin adenine dinucleotide, an electron carrier o Can accept 1 or 2 hydride ions and thus 1 or 2 electrons o Permanently bound to PDH complex – NAD can dissociate and move into the mitochondrial matrix o FAD bound to E3 in the PDH complex, made from Vit B2 o The Dimethlylisoalloxazine ring is what accepts the hydride ions as these have double bonds which

break and hydrogen can be attached

NAD

-‐ Nicotinamide adenine dinucleotide o Carries 1 hydride and 1 electron o Benzoid ring accepts hydride ion (double bond

broken hydrogen can attach) o Sourced from niacin (vitamin B3) o NAD is electron acceptor (oxidized form, NADH is

reduced form as has already accepted a H) o Does not partake in reaction just carries electrons

around the mitochondria – readily recycled o Deficit of Niacin can cause rough skin known as

Pellagra o Niacin is used for the pyrimidine ring in NAD+ o Symptoms: dermatitis, diarrhea, possible death o Niacin is supplemented from tryptophan o Causes: corn based diets (low in tryptophan), alcoholism o Treatment: diet changes

PDH complex

-‐ Each appears in the complex as multiple complexes -‐ Structure helps to control the substrates through the complex -‐ Long arm in Lipoaminde in E2 keeps the substrate in complex controlled, holding intimidate substrates close

to the complex keeps the rate of the reactions to stay rapid as the intimidates don’t diffuse away also makes sure the substrates are available for the reactions and that they are not used by any other enzymes or lost

-‐ Overall reactions sees pyruvate converted to acetyl CoA and 1 carbon dioxide is released, all of the 3 enzymes (E1-‐E3) as well as the 5 coenzymes are needed

-‐ Very favorable as gives a large negative Delta G


PDH COMPLEX REACTION BREAKDOWN 5 STEPS

1. Pyruvate is decarboxylated and product (acetyl group) binds to coenzyme TPP 2. Acetyl group oxidized to acetate and electrons are transferred to thiol groups in lipoamide (reduced) (E2 –

enzyme is Dihydrolipoyl tranacetylase) – 2 carbon acetate binds to the long arm chain of lipoamine via S group.

3. Acetate binds Coenzyme A to make Acetyl-‐CoA (enters citric acid cycle) 4. 2 H-‐ Ions removed from reduced lipoamide (recycled) and transferred to FAD this makes FADH2 (need to

recycle the coenzymes do that PDH can catabolize other pyruvate) E3 is Dihydrylipoyl Dehydrogenase. 5. Electrons transferred to NAD+ (this recycles the coenzyme)

What happens is you have PDH deficit?

-‐ Only anaerobic catabolism of glucose (build up of pyruvate and lactic acid) o Pyruvate cannot be catabolized via citric acid cycle

-‐ Lactic acidosis and PDH deficiency syndrome -‐ Syndrome seen in infancy

o Slow neuronal development and motor skills o Brain requires aerobic catabolism of glucose

-‐ Genetic mutations (mostly E1) -‐ Diagnosis: skin sample and analysis of fibroblast enzyme activity -‐ No treatment

E2 of PDH-‐ clinical study

-‐ E2 of PDH has 2 sulfhydryl groups -‐ Mercury has high affinity for sulfhydryl groups

o Outcompetes and blocks site of enzyme o PDH complex is inhibited

-‐ Mercury used to shape felt hats -‐ Decreased central nervous system function

o This is where “mad as a hatter” came from


Part 2: Citric Acid Cycle (TCA/Krebs Cycle) Contents:

-‐ Central role of citric acid cycle in aerobic energy metabolism -‐ Enzymes, cofactors and metabolic intermediates of the citric acid cycle -‐ Regulation of citric acid cycle -‐ Amphibolic nature of citric acid cycle

Citric Acid Cycle:

-‐ 2 Carbon compound (acetyl CoA) enters the cycle, 2 carbon dioxide atoms are released) -‐ Electrons are transferred to electron carriers NAD and FAD -‐ 1 GTP is made and quickly converted to ATP -‐ Common oxidation pathway for carbohydrates, proteins and fatty acids -‐ Acetyl-‐CoA from the PDH complex enters the cycle

-‐ 8 Different reactions take place in the cycle -‐ Occurs twice to fully oxidize one glucose molecule -‐ 1 Glucose à 2 pyruvate and 2 acetyl CoA

Step 1: Citrate Synthase

Ø Condensation of oxaloacetate (recycled from TCA cycle) and acetyl-‐CoA (combination of 2 molecules, with loss of a small one)

Ø Hydrolysis (addition of water) releases coenzyme A and produces citrate (coenzyme A is reused as a coenzyme in the PDH complex)

Ø Enzyme: citrate synthase Ø Citrate synthase is a dimeric protein (two individual protein) which undergoes conformation changes after

the oxaloacetate bonds, opening up the binding site for CoA – this stops CoA from binding prematurely – otherwise cleavage would see the 2 carbon acetate needed for citric acid cycle would float off

Step 2: Aconitase

Ø Formation of isocitrate (H and OH group swap molecular places in prep for decarboxylation) Ø Occurs through dehydration (water loss) and then hydrolysis Ø Aconitase has an iron III sulfide in the center and helps in substrate binding for citrate Ø Start product citrate and final isocitrate are isomers – this swap occurs with the purpose of allowing further

reactions to occur


Step 3: isocitrate dehydrogenase

Ø Oxidation (loss of an e-‐ transferred to NAD to make NADH) and decarboxylation (removal of carbon) of isocitrate to a-‐ketoglutarate

Ø Isocitrate is oxidized to the intermediate oxalosuccinate – the NADH is what is being reduced). Intimidate stays bound to the enzyme until its decarboxylated and released as a-‐ketoglutarate

Ø Isocitrate dehydrogenase uses manganese ions as a cofactor to stabilize oxalosuccinate

Step 4: a-ketoglutarate dehydrogenase complex

Ø Oxidation and decarboxylation of a-‐ketoglutarate to succinyl CoA Ø Releases CO2 electrons are transferred to NAD to make NADH Ø Identical to pyruvate dehydrogenase complex

o Similar subunits o Same coenzymes

Steps so far: 1. Condensation 2. (a) Dehydration, (b) Hydration 3. Oxidative decarboxylation 4. Oxidative decarboxylation

Products so far:

Ø 2NADH Ø 2CO2

Next steps have the aim of: regeneration of oxaloacetate Part 3: Citric Acid Cycle Part 2 – Regeneration of oxaloacetate Step 5: Succinyl-CoA synthetase

Ø Synthetase: condensation using nucleotide triphosphate (e.g. ATP, GTP) Ø The thio-‐die-‐ster bond highlighted between CoA and Succinyl is a high energy bond – this energy is

harnessed and stored by converting GDP -‐-‐> GTP Ø Released coenzyme is recycled (used in E2 in PDH) Ø Bond between CoA and succinate releases energy which is stored in GTP


o Phosphate group in GTP is transferred to ADP to make ATP uses the enzyme nucleoside-‐

diphosphate kinase Ø The enzyme (succincyl CoA synthetase) has two subunits one binds succinyl CoA and the other the

diphosphate nucleotide

Step 6: Succinate Dehydrogenase

Ø Oxidation of succinate to fumerate using succinate dehydrogenase o FAD is reduced to FADH2

Ø Enzyme contains 3 iron sulfide clusters to help with electron transfer reactions in the electron transport chain

Ø Enzyme is bound to inner mitochondrial membrane

Step 7: Fumarase

-‐ Hydration (add water) of fumarate à malate -‐ Fumerate becomes L-‐Malate -‐ Reaction is reversible -‐ Fumarase is very stereo specific

o Will only interact with Trans form of fumarate, NOT CIS-‐FUMERATE o Will only produce L-‐malate

Step 8: Malate dehydrogenase

-‐ Oxidation of L-‐Malate à oxaloacetate -‐ This step is responsible for the regeneration of oxaloacetate so the citric acid cycle can continue

o Paired with reduction of NAD+ to NADH -‐ Not energy is put in to the reaction – reaction is powered by equilibrium -‐ The concentration of oxaloacetate in cell is quite low however metabolic reactions such as the 1st step in the

citric acid cycle are constantly using oxaloacetate away thereby driving reaction in the forward direction – despite the unfavorable positive delta G


-‐ Reverse reaction more favorable due to high positive delta G

o Oxaloacetate is always in demand which drives reaction to reach equilibrium by creating more Energy conversion/conservation

-‐ Energy is efficiently conserved in the citric acid cycle -‐ Energy is released when molecules are oxidized -‐ 2 carbon acetyl-‐CoA enters and released as 2 carbon

dioxides -‐ Energy released is conserved and stored in reduced

electron carriers (NADH, FADH2) -‐ Reduced electron carriers then proceed on to next system

o Oxidative phosphorylation – makes ATP for cell -‐ Energy conserved by breaking the bond in succincinyl-‐CoA is balanced by converting GTP à ATP TCA cycle is amphibolic and anaplerotic -‐ TCA cycle involves catabolism and anabolism (amphibolic)

o Oxidative catabolism i.e. breakdown of molecules (blue boxes) of carbs, proteins and fats

o Anabolism using intermediates o Oxaloacetate is a precursor for the amino acid aspartate acid

à pyrimidine o Also can be used in gluconeogenesis o Without these, the cell couldn’t make energy efficiently

-‐ Anapleorotic role (red arrows) o Replenishes intermediates of the cycle from external pathways

-‐ Via enzyme PEP carboxylase -‐ The citric acid cycle can only work effectively when there is enough oxaloacetate for the first steps -‐ PEP is regulated positively by acetyl CoA so when it builds up in the cell it indicates the citric acid cycle isn’t

working well enough as there is a build up of the acetyl CoA because there is a low count of oxaloacetate -‐ PEP carboxylase takes PEP directly

from reaction 9 of glycolysis and converts it to oxaloacetate allowing the citric acid cycle to catch up and use the remaining Acetyl CoA

-‐ Then as the levels of Acetyl CoA drop back down, the positive effect of PEP on the citric acid cycle tells it to slow down

Regulation Level 1

-‐ Conversion of pyruvate to Acetyl CoA via the PDH complex

-‐ This is allosterically regulated i.e. there is a direct link to the energy needs of the cells

Negative regulators (turning PDH off) -‐ An abundance of energy (ATP, NADH or

acetyl-‐CoA) and fatty acids (shuts down PDH because acetyl CoA is produced from the beta oxidation of fatty acids so the PDH doesn’t need to function)


Positive regulators (turning PDH on)

-‐ An abundance of AMP of CoA -‐ Indicates the cell is low in energy – needs acetyl CoA therefore the PDH cycle must run

Regulation step 2

-‐ Exergonic reactions that need reactants otherwise the whole cycle is slowed down -‐ Cycle therefore products act as reactants in the next step -‐ AKA rate limiting steps, these are:

o Citrate synthase o Isocitrate dehydrogenase o A-‐ketoglutarate dehydrogenase

-‐ Act as negative inhibitors o Stopped by product inhibition (when products of the enzyme build up in the cell so the enzyme will

shut down – stopping the citric acid cycle) -‐ Calcium is an activator for isocitrate and A-‐ketoglutarate, that is used to indicate the muscles are contracting

meaning the cells require more energy to be produced PDH Complex Regulation

-‐ Control of PDH particular E1 of complex -‐ Allosterically controlled through

phosphorylation -‐ 2 enzymes are used

1. PDH Kinase – phosphorylates (turns off PDH) 2. PDH Phosphatase – dephosphorylates turning PDH on

-‐ When there is plenty of energy (e.g. ADP, NADH, Acetyl CoA) then PDH kinase is turned off phosphorylating E1 turning it off

-‐ When cell needs energy (indicated by NAD, ADP and Pyruvate) PDH phosphatase is turned on which then activates the PDH complex by dephosphorylating the enzyme

-‐ PDH is activated by signals of work e.g. muscle contraction


Clinical Study: Vertebrate Poison 1080

-‐ Poison permanently binds and inhibits aconitase -‐ Shuts down the whole citric acid cycle -‐ Used as a poison to control pest animals -‐ Native Australian species have developed a tolerance for sodium monofluroacetate (immune to poison

1080) Clinical Study – Citrate

-‐ Step 1 of Citric Acid Cycle -‐ Citrate is a metal chelator

o Binds metal ions to inhibit the metal o Citrate synthase produced on a large scale from the fungus aspergillus niger o This is excreted by root cells and builds up those metals in the soil and can be taken up by other

plants that don’t make it themselves o Idea of creating genetically engineered crops to do this o GMO’s increase crop survival and therefore yield

Documents

MB - Lecture 5 - PDH + Citric · Metabolic*Biochemistry* Lecture5* * Lecture 5:The PDH Complex Part 1: Pyruvate Dehydrogenase ! Content:! 4 The!pyruvate!dehydrogenase!reaction!mechanism!!

MB - Lecture 5 - PDH + Citric · MetabolicBiochemistry Lecture5* * Lecture 5:The PDH Complex Part 1: Pyruvate Dehydrogenase ! Content:! 4 The!pyruvate!dehydrogenase!reaction!mechanism!!