metabolism Flashcards
bioenergetics and thermodynamics
Bioenergetics
Quantitative study of energy transduction occurring in living cells.
Study of the nature and function of the chemical processes that are responsible for these energy transductions.
thermodynamics:
First Law states:
“For any physical or chemical change, the total amount of energy in the universe remains constant”
Therefore energy cannot be created or destroyed, but it can be converted from one form to another.
Define the terms exergonic and endergonic
change in =D
free energy change(DG)
Gibbs Free energy (G) is the amount of energy in a system available to do work.
Free energy changes derive from
Changes in heat content ( H) = enthalpy change
Changes in the state of order ( S) = entropy change
DG= DH - T x DS
energy for reactions:
If DG is -ve, energy is liberated and the reaction is said to be exergonic
A reaction canoccur spontaneously only if DG is –ve.
If DG = +ve
the reaction is said to be endergonic
For the reaction to occur it will require an energy input
A system is at equilibrium and no net change can take place if DG is zero.
The DG of a reaction is independent of the path of the transformation.
DG provides no information on the rate of reaction.
State why we need to produce energy in our cells, giving examples
Living organisms require continual input of free energy as many biological processes are endergonic:
Mechanical work e.g. Muscle contraction
Active transport
Synthesis of complex biomolecules from simple precursors
Also Signal transduction (environmental responses), generation of light (fire flies) and electricity (eels)
how is energy derived from the environment by living organisms(plants+animals)
Phototrophs-obtain energy by trapping light
->Photosynthesis
Chemotrophs-obtain energy by oxidation of food stuffs
->Catabolism
chemoorganotrophs
Extract energy from organic compounds by oxidation
Fats 9 kcal/g
Carbohydrates 4 kcal/ g
Proteins 4 kcal/ g
Alcohol 7 kcal/ g
extraction of energy from food
controlled extraction of energy from food:
Regulation and control
Don’t want to release all the energy at once
Don’t want to increase body temperature excessively
Coupled reactions are more efficient
Not very mobile around body
Small carrier molecule better
extraction of energy from food:
Stage 1-Large molecules broken down into smaller units. No useful energy captured
Stage 2-Small molecules degraded into a few simple units that play a role in central metabolism. Some ATP generated.
Stage 3-ATP produced from the complete oxidation of simple units by the final common pathways for oxidation of fuel.
REDOX reactions:
As an organic compound is degraded (oxidised) electrons flow through intermediates to oxygen (the final electron acceptor) or are used to reduce other cellular components.
In a redox reaction
the electron donor is the reducing agent and is oxidised;
the electron acceptor is the oxidising agent and is reduced
Redox reactions involve electron flow which can be made to do work just as they do in an electric circuit.
dehydrogenase and electron carriers
Dehydrogenases oxidise organic compounds by abstracting 2H+ & 2 and passing them to a mobile carrier in biodegradation and energy abstraction ( i.e. respiration).
Dehydrogenases can reduce organic compounds by adding 2 H+ & 2 from a mobile electron carrier typically in biosynthetic pathways
electron carriers:
NADH – produced in catabolic reactions and by TCA cycle
used in the generation of ATP by OxPhos
Usually found inside the mitochondria
NADPH – produced by PPP
used primarily for reductive biosynthesis (e.g. FA synthesis)
Usually found in the cytoplasm
FADH2 – produced in catabolic reactions and by TCA cycle
used in the generation of ATP by OxPhos (generates less energy than NADH)
Usually found inside the mitochondria
Describe the currency of energy transfer in the body
Any living organism must generate ATP to live - when they stop producing ATP they die
ATP is an energy rich molecule with high phosphoryl transfer potential
it contains 2 phosphoanhydride bonds on its triphosphate unit
Free energy (D G ) is negative so thermodynamically unstable.
However kinetically stable (in absence of a catalyst, breakdown is very slow)
For ATP hydrolysis
DG = -7.3 kcal mol-1 or -10.9 kcal mol-1
ATP-ADP cycle:
Fundamental mode of energy exchange in biological systems
ATP principal immediate donor of free energy in biological systems rather than long-term storage form.
Consumed within minutes of formation, very high turnover.
Around 50Kg of ATP consumed in 24h period.
ATP production:
Substrate level phosphorylation
Transfer of phosphoryl group from metabolites with high-phosphoryl transfer potential to ADP producing ATP
Oxidative phosphorylation
Process of ATP formation as a result of transfer of electrons from fuels via electron carriers (NADH or FADH2) to the final electron acceptor oxygen.
In animals over 90% of ATP formed by this method. Carried out in the mitochondria.
metabolism and comparing anabolic and catabolic metabolism
Highly coordinated cellular activity serves four main functions.
Obtain energy e.g. ATP
Convert nutrients into own characteristic molecules
Polymerise monomeric precursors e.g. polysaccharides
Synthesise and degrade molecules required for special cellular functions e.g. intracellular messengers
Hundreds of different enzyme-catalysed reactions.
Central metabolic pathways.
Few in number
Highly conserved throughout nature
Two broad classes of metabolic pathways
CATABOLIC reactions-transform fuels into usable cellular energy
ANABOLIC reactions-utilise the useful energy formed by catabolism to generate complex structures from simple ones.
catabolic:
Degradative
Produces ATP
-ve free energy change
Produces reducing potential
Generates NADH + FADH2
anabolic:
Synthetic
Requires ATP
+ve free energy change
Requires reducing potential
Uses NADPH
Explain why metabolic regulation is required
The human body requires energy to function e.g. breathing, circulating blood, walking etc.
The body does not have a constant external supply of energy.
Energy (food) intake is intermittent usually 3 or 4 times a day.
Yet energy expenditure is continuous (resting metabolism) with occasional extra bursts.
We therefore need to store energy and release it when required.
List the 3 principal ways metabolism is controlled
There are three principal ways by which metabolic pathways are regulated:
->Levels and accessibility of substrates (Thermodynamics and compartmentation)
->Amounts of metabolic enzymes (Rate of transcription and degradation)
->Modulation of catalytic activities of enzymes (Allosteric regulation, Covalent modification, Association with regulatory proteins)
enzyme turnover, modulation of enzyme activity, end product/feedback inhibition, regulatory enzymes
Number of enzyme molecules is a function of the rate of synthesis and degradation, both of which are tightly controlled.
Determined by
Alteration (production) of transcription factor by external signals.
Stability of mRNA species
Rate of translation (dependant on various factors)
Rate of protein degradation
Changes in amount of enzyme present in the cell is relatively slow ranging from minutes to hours.
Metabolic pathways are interdependent
Key enzymes (rate limiting, commitment step) control the flux of substrates through a pathway
These key enzymes can be regulated in a number of ways
end-product:
binds non-covalently to specific regulatory site (allosteric site)
binding is dependent on concn. and binding affinity
induces conformational change affecting active site
regulatory enzymes:
several regulatory sites
each site selectively binds a ligand (activator or inhibitor)
conformation of active site reflects summation of signals
Outline allosteric regulation of enzymes, using adenylate control as an example
Allosteric control:
Allosteric is derived from the Greek meaning “the other”.
An allosteric enzyme has a site distinct from the substrate-binding site. Ligands which bind to this allosteric site are termed allosteric effectors or modulators.
Binding causes conformational changes so affinity for substrate or other ligands change.
Can be Positive (activator) or Negative (inhibitor)
Adenylate control:
Many reactions and pathways in metabolism are controlled by the energy status of the cell.
Energy charge ranges from 0 (all AMP) to 1 (all ATP).
ATP generating pathways are inhibited by a high energy charge.
ATP utilising pathways are stimulated by a high energy charge.
Control of pathways has evolved to maintain energy charge within narrow limits (buffered)
ATP-generating pathways catabolic
e.g. glycogenolysis
glycolysis
b-oxidation
ATP-utilising pathways anabolic
e.g. glycogenesis
gluconeogenesis
lipogenesis
purine + pyrimidine syntheses
Outline covalent modification of enzymes
Modification of existing protein structure by covalent modification is a quicker process than changing the levels of enzyme. (over seconds to minutes)
Various types:
Adenylation
Methylation
Phosphorylation (most common)
Attachment of a functional group covalently to an amino acid side chain, e.g. phosphate
Attachment is selective and enzyme catalysed
Induces conformational change
Phosphorylation/Dephosphorylation tends to alter the conformation of a protein such that:
Changes Vmax and/or Km of the enzyme
Sensitivity to substrate
Sensitivity to inhibitors or activators
Protein “locked” in new conformation
To be useful this must be a reversible process
Generally triggered by an external signal leading to amplification of signal
glycolysis and stages
Ancient pathway employed by a wide range of organisms from the simplest bacteria to humans.
Conversion of glucose to pyruvate.
Does not require O2 (anaerobic)
Located in the cytosol of Eukaryotic cells
Glucose important and common fuel in most cells. In mammals it is the only fuel red blood cells use.
STAGE 1
Trapping and destabilising glucose in order to produce 2 X 3C molecules (5 Steps in the process).
Energy required (2 ATP’s per Glucose molecule)
STAGE 2
Oxidation of the 3C molecules to pyruvate (5 Steps in the process).
Energy generated (4 ATP’s and 2NADH per Glucose molecule)
hexokinase
Can phosphorylate (kinase) a variety of hexose (six carbon) sugars (glucose, mannose even fructose)
Induced fit enzyme action
Equilibrium of reaction strongly favours glucose 6-phospate (effectively irreversible reaction)
Regulatory enzyme of glycolysis, inhibited by glucose 6-P (FEEDBACK INHIBITION)
reversibility and equilibrium of stage 1
Glyceraldehyde 3-P is on the direct pathway of glycolysis. DHAP is not.
DHAP needs to be converted into G 3-P otherwise a 3C fragment capable of generating ATP will be lost.
The enzyme Triose Phosphate isomerase (TIM) catalyses this reversible reaction.
At equilibrium 96% is in the DHAP form. However because of subsequent reaction of glycolysis and removal of Glyceraldehyde 3-P the equilibrium is pushed towards its formation.
triose phosphate isomerase(TIM)
Great catalytic prowess, accelerates isomerisation by a factor of 1010 compared to simple base catalysis
Kinetically perfect enzyme, the rate limiting step is the diffusion-controlled encounter of substrate and enzyme.
So 2 molecules of G-3-P almost simultaneously from F 1,6-bisP
steps throughout stage 1
Step 1: Trapping Glucose
Glucose enters cells via facillitated diffusion through specific transport proteins.
Once in the cell Glucose is trapped by phosphorylation.
Glucose 6-phosphate is negatively charge and cannot freely diffuse out of the cell.
Addition of the phosphate group begins the destabilisation process of glucose, which leads to further metabolism.
Step 2 Formation of Fructose 6-phosphate
Isomerisation of Glucose 6-P to Fructose 6-P is a completely reversible reaction carried out by the enzyme phosphoglucose isomerase.
Convert from one isomer (glucose) to another (fructose) by Tautomerisation
Step 3 is a second phosphorylation reaction.
The enzyme Phosphofructokinase carries out this reaction.
Allosteric enzyme (Tetramer) which sets the pace of glycolysis
Inhibited by ATP, Citrate and H+ ions
Stimulated by AMP, ADP and Fruc 2,6-bisP
Steps 4 and 5: Splitting Fructose 1,6-bisP into useful 3C fragments.
Cleavage of Fructose 1,6-bisP is catalysed by the enzyme Aldolase to yield 2 Triose phosphates
Readily reversible under normal physiological conditions
step 1 summary
Glucose enters the cell via specific transporters.
Phosphorylation of Glucose traps it within the cell and begins the process of destabilisation
The 6C molecule is isomerised from an aldose to a ketose sugar prior to further destabilisation by phosphorylation.
The destabilised 6C sugar then fragments into two interconvertable 3C sugars.
STAGE 1 has utilised 2ATP molecules.
steps throughout stage 2
Step 6: Formation of a High Energy Bond
G 3-P is oxidised and phosphorylated by the enzyme G 3-P dehydrogenase.
Dehydrogenase transfer “high energy” electrons from complex organic molecule to NAD+ to form NADH
Step 7: ATP generation from 1,3-bisPglycerate
Substrate level Phosphorylation
Remember Glucose (6C) yields 2 x 3C intermediates therefore 2 ATP’s generated per glucose molecule.
Steps 8, 9 and 10: Generation of additional ATP and pyruvate formation (2 per glucose molecule)
Phosphoryl group on 3-Pglycerate shifts position, followed by dehydration and formation of a C=C bond.
Increases transfer potential of phosphoryl group.
glyceraldehyde 3-P dehydrogenase and pyruvate kinase
The resulting intermediate 1,3-bisphosphosphoglycerate is an acyl phosphate i.e. has a high-phosphoryl-transfer potential.
Sum of two processes
Oxidation of the aldehyde to a carboxylic acid by NAD+
Joining of orthophosphate to the carboxylic acid
pyruvate kinase
Irreversible transfer of phosphoryl group to form ATP
Substrate level phosphorylation
Regulatory enzyme activated by Fructose 1,6bisP and inhibited by ATP and alanine.
catabolic fates of pyruvate
During glycolysis NAD+ is converted to NADH
Glycolysis cannot continue if [NAD+] decreases.
Oxygen present – electrons on NADH transferred to oxygen (via electron transport chain) to produce H2O, ATP and NAD+.
No oxygen present – Electrons on NADH transferred to pyruvate to form lactate or ethanol and NAD+(recycled for step 6 of glycolysis)
OR
Ethanol Formation: Yeast and some other microorganisms
Anaerobic process (no O2 required)
OR
Lactic acid formation: Microorganisms, also in higher organisms when oxygen is limited e.g. intense exercise
Regeneration of NAD+ for step 6 of glycolysis
Under aerobic conditions much more energy can be extracted by means of the TCA cycle and OX PHOS
Pyruvate enters mitochondria and is oxidised to acetyl CoA.
NADH generated at step 6 of glycolysis cannot enter the mitochondria, so NAD+ is regenerated indirectly by OX PHOS using specific shuttles.
why animals store energy as glycogen / storage of glycogen
Controlled breakdown and synthesis helps maintain blood-glucose levels (Normal 5mM)
Important as Glucose is only fuel for brain under non-starvation conditions
Glucose from glycogen is readily mobilised. Good source of energy for sudden, strenuous activity.
Unlike fatty acids can provide energy under anaerobic conditions
storage:
Two major sites of storage:
Liver (10% by weight)
Muscle (2% by weight)
Insoluble granules in cytosol
Pathways of glycogen metabolism the same in Liver and Muscle
Regulation differs
glycogen synthesis - glycogenesis and its molecular process
Glucose uptake from the blood is facilitated by transport proteins
GLUT 2 present in liver and b cells of pancreas
High capacity, low affinity transporter
takes up a lot of glucose when there is a lot around e.g. after a meal
GLUT 4 present in muscle and fat cells
Insulin leads to rapid increase in number so increasing uptake
Amount of transporters in muscle membranes increased by excercise
molecular process:
Formation of a-1,6 glycosidic bonds in catalysed by a branching enzyme.
Removes 7 glucose unit from end of chain which is a least 11 residues long
Re-attaches it at a more interior site.
Must be at least 4 residues away from a branch point
steps of glycogen synthesis - glycogenesis
Conversion of Glucose to Glucose 6-P
Hexokinase phosphorylates glucose in order to trap it within cell. The product inhibits the enzyme
Liver rich in an isozyme called Glucokinase:
Not inhibited by glucose 6-P
High Km, i.e. affinity 50 times lower than Hexokinase
Role of Glucokinase is to provide glucose 6-P for synthesis of glycogen and formation of fatty acids
Gives brain and muscles first call of glucose when limited, but ensures it is not wasted when abundant
Glucose 6-P is the converted to Glucose 1-P by the enzyme phosphoglucomutase
Active mutase enzyme contains a phosphorylated serine residue
The glucose 1-P is activated by an enzyme UDP-glucose pyrophosphorylase, which produces UDP-glucose
This is a reversible reaction, but the equilibrium is shifted towards UDP-glucose formation by hydrolysis of the pyrophosphate
Glycosyl units added to the non-reducing end of glycogen molecule to form an a-1,4 glycosidic bond.
Reaction catalysed by the enzyme Glycogen Synthase.
A primer is required as the enzyme can only add glycosyl units if polysaccharide chain is greater than 4 residues
Primer function carried out by glycogenin
glycogen synthase
Regulatory enzyme of glycogen synthesis
Regulated by covalent modifications i.e. phosphorylated by the enzyme Protein kinase A
Phosphorylation converts the active a form of the enzyme into a usually inactive b form
Insulin counteracts the phosphorylation and so signals the body to synthesise glycogen (activates phosphatase enzyme)
At high levels Glucose 6-P can allosterically activate of the b form
glycogenolysis
Biosynthetic and degradative pathways rarely operate by precisely the same reactions.
Afford greater flexibility both energetically and regulation
Glycogen breakdown provides glucose 6-P for further metabolism
steps of glycogenolysis
Glycogen phosphorylase cleaves a-1,4 glycosidic bonds by addition of orthphosphate (sequential removal from non-reducing end)
Release of Glucose 1-P (negative charge)
Fully reversible but under physiolgical conditions [Pi]/[Glucose 1-P] is greater than 100
The a-1,6 linkages cannot be cleaved by glycogen phosphorylase (sterically hindered when 4 residues from branch)
A debranching enzyme is required, in eukaryotes this is a single enzyme with two activites, transferase and a-1,6-glucosidase activity
Activity of the debranching enzyme paves the way for further cleavage by glycogen phosphorylase
Glucose 1-P is then converted to Glucose 6-P by phosphoglucomutase
Glucose 6-P is negatively charged so cannot escape from the cell .
Liver because of its role in blood glucose homeostatis has a Glucose 6-phosphatase enzyme located in the ER
Hydrolytically cleaves Glucose 6-P to Glucose which can enter the blood
glycogen phosphorylase
dimeric protein regulated by several allosteric effectors, signal the energy state to the cell
reversible covalent modification
responsive to hormones such as insulin, adrenaline and glucagon
glycogen phosphorylase regulation differs depending on the tissues function
muscle uses glucose itself as a source of energy
liver maintains glucose homeostasis of the whole organisms
Glycogenesis and Glycogenolysis are reciprocally regulated
Synthesis and breakdown are regulated by hormone triggered (adrenaline and/or glucagon) cAMP cascade acting through protein kinase A
role of protein phosphatase 1
Activated by signal cascade brought about by high insulin levels
Reverses effect of Protein kinase A
Inactivates phophorylase kinase and phosphorylase a by removing covalent P group
Activates glycogen synthase by removing covalent P group
diseases linked to glycogen metabolism - glycogen storage disease, Von Gierkes disease, Coris disease, McArldes disease
glycogen storage disease
Inherited genetic defects which mean enzymes needed to degrade stored glycogen aren’t working properly
Von Gierkes disease
most common form (type I)
autosomal recessive, 1 person in 200,000
Glucose-6-phosphatase missing
Hypoglycemia due to lack of liver gluconeogenesis and glycogenolysis
Need regular supply of glucose even when asleep
Coris disease
Type III
Deficiency of Glycogen debranching enzyme
Glycogen accumulates with short side branches as these can’t be utilised by phosphorylase
Much less sever symptoms than von Gierkes disease
McArldes disease
Type V
Muscle Phosphorylase missing
can’t break down stored glycogen in muscle
muscle cramps (due to high levels of ADP and alkaline conditions), can’t perform strenuous exercise
no increase in lactate in muscle upon exercise
indicate the importance of glucose as a fuel molecule and the finite store available in the body
gluconeogenesis - Glucose formation from non-carbohydrate precursors
Carbohydrates are important molecules in biological systems.
Synthesis of carbohydrate containing biological molecules relies on a source of activated monosaccharides.
In some situations these activated molecules are derived from non carbohydrate precursors, e.g seedlings, bacteria, starving mammals.
Major site of Gluconeogenesis is the liver. Kidney during extreme starvation.
Helps maintain blood glucose levels so brain and muscle can extract it.
Converts pyruvate into glucose
NOT THE REVERSE OF GLYCOLYSIS
Precursors first converted to pyruvate or enter pathway further along (at Oxaloacetate or DHAP)
glucose:
Primary fuel for the brain normally GLUCOSE
ONLY fuel for Red Blood Cells is GLUCOSE
Daily requirement for glucose 160g (Brain 120g)
Readily available glucose (sufficient for 1 day)
20g Body fluids
190g Glycogen
What about longer periods of starvation or prolonged exercise?
Recognise the requirement for a route for glucose production from a non-carbohydrate source, Identify the precursors
Lactate – Skeletal muscle when glycolysis exceeds oxidative metabolism. RBC.
Amino acids – Diet or during starvation (Muscle breakdown) NOT LEUCINE or LYSINE
Glycerol – Hydrolysis of TAG yields glycerol and fatty acids
Outline the gluconeogenic pathway and identify the key regulatory enzymes - Bypass 1: pyruvate to PEP
Two step process
Step 1: Carboxylation of pyruvate to oxaloacetate by Pyruvate Carboxylase
ANAPLEROTIC Reaction (“Fill Up”)
Step 2: Decarboxylation and phosphorylation of oxaloacetate by Phosphoenolpyruvate Carboxykinase.
GTP required (donates the phosphate group)
Enzyme located both in cytosol and mitochondria
Mitochondrial Phosphoenolpyruvate Carboxykinase used if lactate is glucogenic precursor (Lactate to Pyruvate yields NADH)
Cytosolic Phosphoenolpyruvate Carboxykinase used if pyruvate is glucogenic precursor. (Used if reducing equivalents low i.e NADH needed)
Mitochondrial Phosphoenolpyruvate Carboxykinase used if lactate is glucogenic precursor (Lactate to Pyruvate yields NADH)
The NADH generated in the cytosol is used for the conversion of 1, 3 bisPglycerate to glyceraldehyde 3 phosphate by the enzyme Glyceraldehyde 3-phospahte dehydrogenase further up the pathway
Cytosolic Phosphoenolpyruvate Carboxykinase used if pyruvate is glucogenic precursor. (Used if reducing equivalents low i.e NADH needed)
Oxaloacetate cannot directly diffuse out.
Converted to Malate which leaves via specific transporter.
Malate converted back to Oxaloacetate with concomitant production of NADH (required later)
Outline the gluconeogenic pathway and identify the key regulatory enzymes - Bypass 2: Fructose 1,6-bisP to Fructose 6-P
PEP is metabolised by the enzymes of glycolysis but in reverse until Fructose 1,6-bisP is formed.
The reactions are near equilibrium so when conditions favour gluconeogenesis they will be driven in the direction of Fructose 1,6-bisP
PFK catalyses an irreversible step likewise Fructose 1,6-bisP to Fructose 6-P is irreversible
The enzyme responsible is Fructose 1,6-bisphosphatase which is an allosteric enzyme that catalyses the hydrolysis of the C1 phosphate group
Fructose 1,6-bisphophate +H2O fructose 6-phosphate + Pi
Outline the gluconeogenic pathway and identify the key regulatory enzymes - Bypass 3: Glucose 6-P to Glucose
In most tissues conversion of Fructose 6-P to Glucose 6-P is the end of Gluconeogenesis
However tissues responsible for maintaining blood glucose homeostatis (Liver and Kidney) need to convert glucose 6-P to Glucose
Muscle cannot directly increase blood [glucose]
Takes place in ER
Discuss the reciprocal regulation of glycolysis and gluconeogenesis particularly in the liver
Equilibrium of glycolysis lies far in the direction of Pyruvate production
Mostly due to the three irreversible reactions (Hexokinase, PFK and Pyruvate Kinase)
These reactions must be bypassed during Gluconeogenesis
cori cycle and lactic acid production
Lactate formed by active muscle is converted to glucose by the liver
lactic acid production:
Vertebrates are mainly aerobic organisms (pyruvate is completely oxidised to CO2 and water).
During extreme muscular activity oxygen delivery to muscle is lower than oxygen requirements for oxidation of NADH.
NADH is oxidised by transfer of electrons to pyruvate to form lactate.
Lactic acid dissociates to lactate and H+.
The pH (-log[H+]) therefore decreases.
Muscle pain and failure to contract.
Activity decreases
“Oxygen debt” reduced in about 30 mins by conversion of lactic acid to glucose by gluconeogenesis in the liver.
link reaction and its cofactors
- Irreversible
- Pyruvate dehydrogenase is large complex
– Mass 4 million to 10 million daltons
– Groups travel from one subunit to another connected to the core by tethers
cofactors:
* Catalytic cofactors
– Thiamine pyrophopshate (TPP)
– Lipoic acid
* Stoichiometric cofactors
– CoA
– NAD+
link reaction steps
Pyruvate Dehydrogenase Component (E1)
1. Decarboxylation
* Pyruvate combines with TPP before decarboxylation
* TPP is the prosthetic group of the enzyme
- Oxidation
* Hydroxethyl group is oxidised & transferred to lipoamide
* Forms energy rich thioester bond
Dihydrolipoyl Transacetylase (E2)
3. Group transfer – acetyl group
– acetylation
* Acetyl CoA
– Fuel for the TCA
Dihydrolipoyl Dehydrogenase (E3)
4. Oxidation of dihydrolipoamide back to lipoamide
* Must occur before further acetyl CoA can be formed from pyruvate
* Eletrons transferred to FAD then to NAD+
regulatory point of link reaction and disease examples
Committed irreversible step – ATP/Lipid/aa formation
Pyruvate Dehydrogenase – Selected mechanisms Adenylate control (E1):
- ↑ADP&Pyruvate-PDP
- ↑ATP-PDK
- ↓NADH
- ↓AcetylCoA
- ↑Ca2+
diseases:
Mercury
– Binds pyruvate dehydrogenase (E3)
– Inhibits enzyme
– Hatters used mercury nitrite
– Sulfhydryl treatment
Beriberi
- alcoholics
– B1 deficiency (thiamine)
– Neurological and cardiovascular symptoms – Raised blood pyruvate levels
– Nervous system relies on glucose for energy
citric acid/krebs cycle
- Final common pathway for fuel oxidation
– Carbohydrates
– Fats
– Amino acids - Most enter at acetyl coenzyme A (Acetyl CoA)
– All enter as components of the TCA
enzymes involved in citric acid cycle
citrate synthase:
* Aldol condensation followed by hydrolysis
* 4C + 2C = 6C
* Entry point for Acetyl CoA
aconitase:
* Isomerisation (dehydration-hydration)
* Rearranges hydroxyl
– Necessary for oxidation step to follow
* Aconitate intermediate
Isocitrate dehydrogenase:
* Oxidation – Reduction & Decarboxylation
–NAD+ →NADH+H+
– CO2
* Oxalosuccinate intermediate
isocitrate dehydrogenase as a regulatory point:
* The first enzyme to generate high energy e-
* Isocitrate Dehydrogenase
– ↑ADP - allosteric
– ↓ATP – allosteric
– ↓NADH – competitive – product
* Excess citrate build may inhibit PFK
ɑ-Ketogluterate Dehydrogenase:
* Decarboxylation – Oxidation – Group Transfer
* An enzyme complex
* Homologous to pyruvate dehydrogenase
ɑ-Ketogluterate Dehydrogenase as a regulatory point:
* The 2nd enzyme to generate high energy e-
* α-ketogluterate dehydrogenase
– ↓Succinyl CoA – competitive – product – ↓NADH – competitive – product
– ↓ATP
* Excess substrate can be used to make aa’s
Succinyl CoA Synthetase:
* Succinyl CoA has a high energy thioester bond
* Conversion to succinate coupled to GTP formation
* Nucleoside diphosphokinase
* GTP + ADP GDP + ATP
* Group transfer – phosphoryl group
next steps of kreb cycle: Oxidation – Hydration – Oxidation
enzymes involved
- Reforms oxaloacetate
- Common series of reactions
– Fatty acid oxidation
– Fatty acid synthesis
– Amino acid breakdown
Succinate Dehydrogenase
* Hydrogen acceptor is FAD
– FAD → FADH2
– Does not dissociate from enzyme
– Passed directly to coenzyme Q
* Forms direct link with electron transport chain
Fumerase
* Addition of H+ and OH-
* Removal of double bond
Malate Dehydrogenase
* Positive ΔG
– Driven by product use in ETC & TCA cycle
the glyoxylate cycle
- Carbs from lipids
- 2x Acetyl CoA
- Succinate – To TCA
- Plants
– Glyoxomes - Some Bacteria
- Oil rich seeds
