Module 8 - Citric Acid Cycle + Acetyl-CoA Flashcards
What is aerobic respiration and what process does it involve
Most important process to produce energy for a cell
Involves: glycolysis, pyruvate oxidation, the citric acid cycle and electron transport
○Sugars, fats and protein enter these pathways and can break down to produce ATP energy for an cell
What is the citric acid cycle and where does it happen,
- aka the tricarboxylic acid cycle
- takes place in the matrix or fluid of the mitochondria where mitochondrial DNA is found and where fatty acid breakdown takes place
- a series of eight rxns break down the two-carbon acetylCoA to the one-carbon molecule of CO2, while at the same generating three molecules of NADH, one molecule of FADH2 and GTP
How many chemical reactions take place in citric acid cycle, what are the products and reactants
● Involves eight chemical reactions that use acetyl coA and oxaloacetate to produce carbon dioxide, NADH, GTP and FADH2
Give a quick overview of Citric acid cycle
Step 1: 2C molecule and 4C molecule combine to form 6C molecule which undergoes biochemical changes an at end original 4C molecule is produced
- Each time C is lost CO2 is released
- 2 CO2 are formed during conversion of 6C to 4C
What is the 2C molecule and how is it formed. Where is it formed? What are the products?
● The 2-carbon molecules come from pyruvate (product of glycolysis in the cell, transported into the mitochondrial matrix where it is oxidized to acetyl CoA by pyruvate dehydrogenase → produces one NADH and one CO2 for each of the two pyruvate molecules made from glucose.)
What does the citic acid cycle start w/
●Acetyl CoA is the starting point of the cycle, the acetyl group from acetyl CoA is transferred to oxaloacetate from citrate.
How many reactions until succinate is formed and what does it produce by then
●4 different rxns lead to the formation of succinate
○Produce two co2, two NADHS and one ATP
What is Succinate recycles back into and how many rxns does that take and what does it produce during that time
● Succinate is then recycled back to oxaloacetate through three more reactions = produce FADH2 and one more NADH = important for fully functioning cells
What are the two stages by which pyruvate is oxidized
○1st the conversion of the three-carbon pyruvate to a two-carbon molecule of acetylCoA,
○2nd the oxidation of acetylCoA to carbon dioxide through the citric acid cycle
How does pryruvate become acetylCoA
● The oxidation of pyruvate to acetylCoA is catalyzed by the enzyme, pyruvate dehydrogenase (PDH) which is located in the mitochondrion
What happens to NADH and FADH2. What does this do to the energy
● All reducing equivalents NADH and FADH2 will go on to the final stage of metabolism which is the electron transport chain which reduces the potential energy
What is the strucutre of the mitochondria
- has double membrane, outer mitochondrial membrane surrounds the entire structure while the inner mitochondrial membrane is invaginated to increase the surface area of the membrane
- invaginations are called cristae and the inner compartment is called matrix
- Space bwn the inner and outer membrane is called intermembrane space
- outer mitochondrial membrane is highly permeable with large channels that span the membrane are called porins which allow compounds less than approx 5000 daltons in size to pass
- the inner mitochondrial membrane is relatively impermeable, allowing only small uncharged compounds like CO2 and water to cross the membrane
How do larger or charged molecules cross the inner membrane
○Larger or charged molecules like pyruvate, protons or ATP will cross with transport proteins
Describe the inner membrane compared to the outer membrane of the mitochondria
- inner contains a much higher concentration of proteins as it contains these specific transport proteins as well as the protein complex involved in the electron transport chain,
○the relative permeability of the inner mitochondrial membrane allows a proton gradient to be established for the electron transport chain
Where are enzymes for citric acid cycle located
Inner membrane, Matrix
What does the Matrix of the mitochondria contan
-PDH pyruvate dehydrogenase complex Citric acid cycle enzymes - Fatty B oxidation enzymes -AA oxidation enzymes -Many other enzymes -ATP, ADP, PI, Mg, Ca, K
What is Coenzyme A.
What partof it is reactive
- a coenzyme that participates in the pyruvate dehydrogenase rxn, it is a derivative of pantothenic acid, which is a B vitamin
- Free thiol group is the reactive part of coenzyme A which will form an energy-rich thioester bond with the two-carbon acetyl group that will be derived from pyruvate
- In reduced form it is also called CoASH
What is Coenzyme A
- a coenzyme that participates in the pyruvate dehydrogenase rxn, it is a derivative of pantothenic acid, which is a vitamin B
- Free thiol group is the reactive part of coenzyme A which will form an energy-rich thioester bond with the two-carbon acetyl group that will be derived from pyruvate
- In reduced form it is also called CoASH
- functions as a carrier that serves to activate the two-carbon acetyl group through the formation of that high-energy thioester bond
What is Pyruvate Dehydragenase,
What does it do
- Stage 1
- Catalzyes the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA
- Oxidative: because electrons are reducing NAD+ to NADH
- decarboxylation reaction because a one-carbon carbon dioxide molecule is removed from the three carbon pyruvate.
What is the leaving group in pyruvate that makes it a 2C acetyl group
carboxyl group of pyruvate is good ‘leaving group’ which leaves in the form of carbon dioxide
What does CoASH attach itself to and what is its fxn
● Free thiol group from CoASH is forming that energy-rich thioester bond (in red) with two-carbon acetyl group derived from pyruvate
- effectively activating the 2C acetyl group
What is the structure of Pyruvate Dehydrogenase and what else does it require
- multi-enzyme complex Consists of 3 Core enzymes and 2 Regulatory enzymes
- three core enzyme subunits, named E1, E2 and E3, which participated in the oxidative decarboxylation of pyruvate
- Two regulatory enzymes: PDH kinase (inactives PDH by phosphorylation) and PDH phosphase (activates PDH by dephosphorylation)
- ALSO REQUIRES: 5 Co-factors
Whats so important about the E2 enzymes-
Contains Lipoic acid which is an important coenzyme that is covalently attached to E2 enzyme that serves as a ‘swinging arm’ for the acetyl group as it goes from one enzymatic rxn to the next in the overall rxn of the pyruvate dehydrogenase complex.
What are the names of the 3 core enzymes
E1: Pyruvate dehydrongenase
E2: Dihydrolipoamide transacetlase
E3: Duhydrolipoamide dehydrogenase
What are the 5 Coenzymes required for PDH and what are their functions
○NAD+ electron carrier
○FAD+ electron carrier
○Thiamine pyrophosphate (TPP) coenzyme of pyruvate decarboxylase has two thiol groups that can undergo reversible oxidation to a disulfide bond
○Lipoic acid: serves as electron (H) carrier and acyl carrier
○CoA serves as a carrier of the activated acyl group
What are the advantages of Multienzymes complex
- Increased rate or efficency of rxn due to minimized diffusion distance for intermediates between enzymes
- Minimized side reactions between successive enzymes in the pathway (In PDH this is lipoic acid)
- Rxns catalyzed by multienzyme complexes can be coordinately regulated
What does PDH kinase and PDH phophatase do
Kinase phosphorylated - bring PDH to its inactive PDH-b form
Kinase phosphatase - bring PDh to its active PDH-a form
PDH kinase is allosterically regulated by:
Acetyl-CoA, ATP, and NADH which activated PDH kinase and promotes phosphorylation
-●Elevated level of acetylCoA and NADH signal to PDH that they have accumulated in the call and therefore, there is no need to further breakdown pyruvate until they can be utilized further downstream.
●Elevated levels of ATP signal that the energy status of the cell is high and that no further pyruvate breakdown is necessary.
- Negactive allosteric regulator
PDH phosphatase is allosterically regulated by:
Insulin and Ca2+, activates PDH phosphatase to promote dephosphorylation
- insulin is released following the consumption of a meal, signals that glucose is abundant and can be broken down for energy and any excess glucose can be converted to acetylCoA which is the precursor for fat synthesis.
○Through this mechanism, excess glucose will be converted to fat for longer-term storage
●Ca2+ is released during exercise to signal muscle contraction
●By activating PDH phosphatase, it will promote activation of PDH to its active form to increase flux of pyruvate into the citric acid cycle for the continued generation of ATP to support the exercising muscle.
●Balance of positive and negative factors will determine whether PDH exists in an active a-form or less active b-form
What does elevated levels of pyruvate, ADP and NAH+ do
inhibit the kinase and therefore promote the active form of PDH and PDHa.
What does competitive inhibition look like in regards to PDH
- regulated by feedback inhibition when concentrations of acetyl-CoA and NADH are elevated.
●These serve to feedback and inhibit PDH when they accumulate in the cell.
What is the net reaction of the citric acid cycle and the equation
3 NADH, 1 FADH2, 1 GTP, 2 CO2m and free coenzyme A
2NAH+ + FAD + GDP +Pi + acetyl CoA ~ 3NADH + FADH2 + GTP + CoA + CO2
How many enzymes are used in the citric acid cycle
● Are 8 eight enzymes in the citric acid cycle and all of these are compartmentalized in the mitochondria.
What about all the enzymes of the citric acid cycle and what are the exceptions
●All citric acid cycle enzymes are soluble within the matrix, with exception of the enzyme, succinate dehydrogenase, which is a member of protein that is contained in the inner mitochondrial membrane
What is the origin of the name citric acid cycle
●Named after the product of the first reaction, citrate
●Citrate is a tricarboxylic acid
●cycle because it begins and ends with oxaloacetate
How are NADH, FADH2 and pyruvate regenerated
though the electron transport chain
What are the three ways regulatory enzymes of the citric acid cycle are regulated:
○Availability of substrates, oxalate and acetylCoA must be available in 1:1 ratio for this first rxn also there must be an adequate supply of NAD+ to allow the cycle to continue
○Competitive inhibition by-products: accumulation of products such as NADH will inhibit some of the key enzymes in the pathway such as the dehydrogenases that are responsible for generating these reducing equivalents
○Allosteric regulation, some key enzymes are subject to positive and or negative allosteric regulation.
- Ex. Ca+ positive regulation, ATP negative regulation, ADP positive regulation
What are common themes in metabolic regulation
1) Energy status of the cell, reflected by the relative concentrations of ADP and ATP
2) Redox state of the cell, reflected by the relative concentrations of NADH and NAD+ are important regulators of metabolism
3) Regulatory enzymes upstream in pathway to be subjected to feedback inhibition by products, serves as a fine-tune flux through the pathway overall
○ Not every enzyme in a pathway is regulated, but there are normally two or three key enzymes in a pathway that represents points of regulation
What does it mean for the citric acid cycle to be AMphibolic
it is both catabolic and anabolic
What happens during the catabolic process of the citric acid cycle
- : oxidavely breaks down glucose fat and amino acids, producing energy & break down produces starting material for the biosynthesis of some compounds
- Citrate is siphoned off to produce fatty acids and steroids
- succinylCoA is siphoned off for the synthesis of heme and chlorophyll.
- Alpha-ketogluarate and oxaloacetate are siphoned off for the synthesis of some amino acids as well as purines and pyrimidines, respectively
What happens during the anabolic process of the citric acid cycle
- citric acid cycle generates reduced factors, such as NADH and FADH2.
- citric acid cycle intermediates can also be starting materials for biosynthetic pathways
What is anaplerosis and what is the most important anaplerotic reaction
○The process of replenishing citric acid cycle intermediates that have been extracted for biosynthesis
- Most important anaplerotic rxn is Synthesis of oxaloacetate from pyruvate
What are processes that are involved in the anaplerotic pathway in the citric acid cycle
●Pyruvate carboxylase catalyzes the following reaction: pyruvate + Co2 + ATP + H2O→ oxaloacetate + APP + P
Prruvate + malic enzyme → Malate
What ensures a 1:1 ratio of oxaloacetate:acetylCoA
● If the supply of acetyl-CoA (form glycolysis or B-oxidation) is greater than the supply of oxaloacetate, then citrate synthase will not function maximally due to substrate limitation.
● Therefore, excess acetyl-CoA will inhibit PDH, diverting pyruvate to the pyruvate carboxylase rxn.
What is the Kerbs cycle neumonic, what does each mean
Can I Keep selling sex for money officers The products of each step chronologically -Citrate -isocitrate -a-ketoglutarate. -succinyl CoA -succinate -fumarate -malate -oxaloacetate
What happens in the first step of Kerbs cycle
- aldol condensation
- an enzyme called citrate synthase joins the two-carbon acetyl group from acetyl coA with the four-carbon oxaloacetate to form a six-carbon citrate
- highly exergonic (negative delta G/standard free energy) due to the high-energy thioester intermediate
- essential because oxaloacetate is normally very low (micromolar).
What is the second step of the Kerbs cycle
- dehydration and rehydration
- an enzyme called aconitase converts citrate into isocitrate
- Aconitase contains an iron-sulfur center which acts both in the binding of the substrate at the active site and in the catalytic addition or removal of H2O.
What is the 3rd step in Kerbs Cycle
- Oxidative Decarboxylation
- isocitrate dehydrogenase enzyme oxidizes isocitrate, a six-carbon molecule to a five-carbon a-ketoglutarate.
- The carbon that was lost is released as carbon dioxide and one NADH is also formed
- Mn2+ in, active site stabilizes
What is the 4th step in Kerbs Cycle
- Oxidative decarboxylation
- an enzyme called a-ketoglutarate dehydrogenase converts a-ketoglutarate into a four-carbon succinyl CoA
- Produces 1 CO2 and 1 NADH
What is 5th step in Kerbs Cycle
- substrate level phosphorylation
- succinyl coA synthase enzyme converts succinyl CoA into succinate, produces GTP which is converted to ATP (or vis-versa)
- Like acetyl CoA has thioster bond w/ large negative free energy of hydrolysis. Energy released in breakage of bond used to make GTP. Succinate formed in process
What is the 6th step in Kerbs Cycle
- Dehydrogenation
- an enzyme called succinate dehydrogenase converts succinate to fumarate
- Makes one FADH2
What is the 7th step
- hydration
- a fumarate hydratase enzyme then converts fumarate to malate
What is the last step in kerbs cycle
- Dehydrogenation
- malate dehydrogenase enzyme converts malate back to oxaloacetate
- NADH is produced
Where do we get fat from
from consuming too many calories from sugar, fat or protein
WHat are sources of acetyCoA
Pyruvate breakdown, breakdown of fats (triacylglycerols), ketone bodies and some amino acids
What is structure of triglucerides, what do they do in our body
●Fats are stored in the body as triacylglycerols (aka triglycerides)
●Triglycerides made of 3 fatty acids that have been esterified with glycerol
Usually made of 2 or more types of fatty acids
Where is fat stored
found in adipose tissue in specialized cells called adipocytes
- some muscle also as small amounts of fat
Where are triglycerides broken down
In adipocytes
How are triglycerides broken fown
● Lipase is enzyme which releases the free fatty acids from the glycerol moiety by hydrolyzing the ester bonds
What causes lipase to be stimulated
●When glucose levels are low, hormone glucagon is released and stimulates the lipase to breakdown fats to provide energy
●Beta oxidation allows free fatty acids will be broken down and oxidized to generate reducing equivalents NADH, FADH2 and acetylCoA, which feeds into the citric acid cycle to make energy in the form of GTP and even more reducing equivalents
Where do fatty acids go when theyre NOT beta oxidized
● Fatty acids from lipase leave the adipose tissue and enter the bloodstream to be delivered to other tissues
● Fatty acids are carried through the bloodstream bound to protein albumin, which keeps fatty acids soluble
What happens to fatty acids that get takedup into tissue
● Fatty acids taken up into tissues are broken down to acetylCoA via beta oxidation pathway
What happens to the glycerol from triacylglycerol
● The glycerol moiety is also unstable energy and leaves the adipose tissue to be taken up by liver which is where gluconeogenesis takes place (converted into glucose)
● Glycerol is a substrate for gluconeogenesis entering the gluconeogenic pathway at midpoint
Where in body are fatty acids used
preferred fuel for muscles at rest
Liver uses fat for energy
What must happen before fatty acid enters beta oxidation pathway
Must be activated
How are fatty acids activated
- Attaching attaching a long chain hydrocarbon of fatty acid to coenzymeA
- similar to pyruvate becoming acetyl CoA
- Now acyl CoA is ready to be transported into mitochondrial matrix to be oxidized
Where are enzymes of beta oxidation located and where does it occur
● Enzymes of beta oxidation are all located in the matrix
What happens during Beta Oxidation of Fatty acids
● One round of beta oxidation involves sequential removal of two carbons at a time from the acyl group
- Cleaving bond involves series of four reactions that occur in single round beta oxidation
●you make 1NADH and 1 FADH
●Beta oxidation since it cleaves the carbon-carbon bond adjacent to beta carbon
●Then shortened acylCoA starts the process again entering a subsequent round of beta oxidation and cleaves two more carbons
●The process reapers until all of the acyl group is cut into two carbon acetylCoAs (for even-numbered fatty acids)
What is an alternate fate for AcetylCoA then the citric acid cycle
under starvation → ketogenesis
●Convert fatty acids to ketone bodies in the liver and during fasting or starvation with low glucose since bodies glycogen stores are depleted, can use ketone bodies for energy in brain
What happens to Ketone bodies during fasting
during faster enter bloodstream and cross blood-brain barrier and be taken up by brain and broken back down into acetylCoA which can enter citric acid cycle and produce energy
Does anabolic fat synthesis share any of the same enzymes
- No unlike glycolysis and gluconeogenesis
What happens conceptually during fat synthesis
the process is reverse of beta oxidation
● Two carbons originating from acetylCoA are added one at a time to elongating fatty acyl chain
When do bodies synthesize fat
- Bodies are in mode of synthesizing fat when we have excess intake of carbohydrates (replenish stores then excess broken down via glycolysis and pyruvate dehydrogenase rxn to make acetylCoA) → where acetyl CoA originates from
What does the anabolic process of fat synthesis involve
- Anabolic process = reductive → we have to inject or donate e= into newly synthesized compound (in this case fatty acid)
Where must acetyl CoA be for fat synthesis to take place and how does it get there
- Tricarboxylate transport system must remove acetylCoA via citrate intermediate out of mitochondrial matrix into cytosol (that’s where fat synthesis takes place)
What does fatty acid synthetase do
catalyzes synthesis of fatty acids
What do enzymes elongases and desaturases do
Elongase - can make longer chain fatty acids
desaturases - create double bonds in unsaturated fatty acids
What happens once fatty acids are synthesized
they are esterified with glycerol to form triacylglycerols used to make membrane lipids
What happens to fatty acid metabolism after a meal
●Insulin activates fat synthesis in the liver and simultaneously inhibits fat breakdown in adipose tissue
●Futile cycling: we want to avoid breaking down fats when glucose is plentiful and body is in mode of synthesizing fats
What happens to fatty acid metabolism during periods of low glucose
● Glucagon released during low glucose which is insulins counter hormone
● It stimulates fat breakdown by stimulating lipase in adipose tissue
● Then fats broken down to acetylCoA used for energy or acetylCoA diverted to ketone body synthesis (although not ideal)
What else is AcetylCoA a precursor for
for cholesterol synthesis and synthesis of some amino acids
What does HMG-CoA reductase do
help lower cholesterol levels in those afflicted with high levels of “bad cholesterol”, LDL
Can amino acids made AcetylCoA or pyruvate
Yes certain amino acids can become acetyl CoA while others can become Pyruvate by either entering the citric cycle or directly becoming pyruvate or acetyl CoA
what does Aconitase do
- an enzyme called aconitase converts citrate into isocitrate
- Aconitase contains an iron-sulfur center which acts both in the binding of the substrate at the active site and in the catalytic addition or removal of H2O.
