Topic 7 - Metabolism Flashcards
What is metabolism?
Metabolism –> chemical interconversion in biological systems
- Occurs through a series of enzyme-catalyzed reactions that constitute metabolic pathways.
What is the essential principle of metabolism?
Precursor ->->->->->-> product
Between precursor and product, you have metabolic intermediates which are generated by small steps (Specific chemical changes).
What are the important conditions needed for metabolism?
Reactions must be specific and condition + entire set of reactions must be thermodynamically favourable (doesn’t mean all reactions are favourable).
What are the three main ways that metabolic pathways are controlled?
Cells/organisms change the rate of pathways in order to meet their needs –> occurs as a result of environmental change.
3 main way
- Change the amount of enzyme –> does happen but is a slow process as you have to change the rate of transcription.
- Change the rate of activity of the enzyme –> allosteric inhibition/activation –> acts quickly. Or covalent modification –> fast but not as quick as allosteric (phosphorylation).
- Changing the availability of the substrate –> increase in substrate –> faster rate.
What is allosteric regulation?
Allosteric enzyme undergoes a conformational change when a regulatory molecule binds –> Binds at a site away from the active site –> this conformational change changes the shape of the active site –> Increases/decreases the affinity of the substrate for the active site.
Difference between anabolism and catabolism?
Anabolism is the building of larger molecules from smaller substrates.
Catabolism is the breakdown of larger molecules to form smaller ones.
Note:
- Most cells do both types of pathways
- They are NOT the reversal of each other
- Use different sets of enzymes
- Often occurs in different cellular compartments.
Example of hydrolysis and dehydration reactions?
Hydrolysis –> splitting with water
Dehydration/condensation –> Making bonds using water
Summary of Oxidation/Reduction reactions?
Oxidation –> Loss of e-
Reduction –> Gain of e-
Summary of Isomerisation reaction?
Isomerisation –> reaction whereby the types of atoms don’t change but their arrangement does.
What is a C-C cleavage reaction?
Cleavage –> splitting reaction
What is a group transfer reaction?
The transfer of one group from one molecule to another molecule –> usually nucleophilic –> all enzyme catalyzed.
What is the main function of glycolysis and is it an important pathway?
Main function –> energy conversion pathways present in all organisms.
When O2 is and isn’t present –> glycolysis is not that important but…
It is essential in red blood cells (not mitochondria) and muscles that contract quickly during intensive exercise –> low O 2
What is the role of the Krebs cycle in anabolism?
- Provides building blocks –> intermediates in the cycle act as precursors for macromolecules.
For example:
Citrate –> fatty acid and steroids
OAA –> amino acids
Succinyl CoA –> porphyrins haem.
Can all molecules receive in the diet enter the Krebs cycle?
Yes every molecule in the diet can act as a source of energy –> final common pathway for oxidation of all fuel molecules.
How is glycolysis regulated?
Via allosteric inhibition and activation
Glycolysis responds to the ratio of ATP to AMP in the cell.
High ATP –> decreased glycolysis
How is the TCA cycle regulated?
- Also regulated allosterically
Effect of phosphorylation on enzyme activity?
Phosphorylation can make enzymes more or less active.
- Phosphorylation takes place on Ser and Thr residues (-OH)
Explain the regulation of pyruvate kinase in the liver.
Pyruvate kinase reaction –> last reaction of glycolysis
Converts PEP to pyruvate
Dephosphorylated form –> more active
Phosphorylated form –> less active
What is the structure of glycogen?
Glycogen is composed of 1,4 glycosidic bonds (forms straight chains as well as 1,6 glycosidic bonds which forms branches.
Why can glucose not be stored in cells?
- Free glucose results in an increase in osmotic pressure as it draws water into the cell –> cell swells –> eventually will undergo lysis.
How is glycogen stored in cells?
Glycogen forms granules –> made of roughly 50,000 glucose residues but has 20,000 ‘ends’ (highly branched structure) –> the large number of ends is important as it allows glycogen to be readily metabolized and mobilized.
Acentrecenter of the granule one finds a protein called glycogenin.
Explain the process of glycogen synthesis (creating a straight chain)?
A nucleotide sugar donor (UDP) provides glycogen chain with glucose (glucose needs to be activated in order to allow the reaction to occur) –> glucose is added to the carbon 4 on the non-reducing end —> formation of a 1,4 glycosidic bond.
This is catalyzed by the enzyme glycogen synthase –> adds GLC to non-reducing end.
Explain the process of glycogen branching?
Requirement –> at least 11 residues in the straight chain –> Becuase 6-7 residues are transferred by glycosyl 4-6 transferase at a time to form a branch and it can only act at a point which is at least 4 residues from the core.
Enzymes breaks 1,4 glycosidic bond and transfer chain to form a new branch via the formation of a 1,6 glycosidic bond.
- Results in the formation of 2 non-reducing ends for glycogen synthase to act on (Requirement –> a preformed alpha 1,4 glucose chain with at least 8 residues).
What is glycogen?
Glycogen is a storage form of glucose –> easily mobilized which means that glucose can be obtained from glycogen easily.
Why do we need to store glucose in the form of glycogen?
- Some tissues can’t degrade fats (fatty acids).
- Brain needs glucose –> fatty acids don’t pass the blood-brain barrier easily.
- Red blood cells need glucose –> degradation of fatty acids needs O2 (Mitochondria) –> but they don’t have mitochondria.
- Rapidly contracting muscle requires glucose.
Explain the process of UDP-glucose formation.
- Glucose + ATP –Hexokinase–> Glucose-6-P + ADP
- Glucose-6-P —Phosphoglucomutase–> Glucose-1-P
- ATP + UDP –> ADP + UTP
- Gucose-1-P + UTP –UDP Glucose phosphorylase–> UDP-Glucose + PPi
Explain the process of glycogen breakdown.
Glycogen phosphorylase cleaves the 1,4 glycosidic bond at the non-reducing end –> phosphorolytic cleavage (different form of hydrolysis) –> adds inorganic phosphate to carbon 1 of leaving glucose.
The enzyme uses a cofactor called pyridoxal phosphate –> comes from vitamin B6 –> phosphate group is involved in acid-base catalysis.
Explain the initiation of glycogen synthesis by glycogenin.
Note –> remember that glycogen synthase can only add to a chain already containing 8 residues. Glycogenin helps solve this.
Glycogenin has 2 enzyme activities
- Glucosyltransferase activity –> uses it active Tyr 194 residues in order to bond with glucose on UDP-glucose
- Chain extending activity –> can add another glucose residues until there are at least 8 residues –> primer needed for glycogen synthase activity.
Explain the process of glycogen breakdown (de-branching).
- Glycogen phosphorylase that removes glucose molecules via phosphorolytic cleavage (only 1,4) –> phosphorylase can act only until 4 residues from the branch point/protein core.
- Then these 3 residues of the remaining 4 are moved to another nearby non-reducing end (transferase activity of debranching enzyme).
- The last glucose with the α 1-6 linkage is hydrolyzed using the debranching enzyme –> frees glucose molecule
- This leaves an unbranched polymer with α1-4 linkages for further phosphorylase activity.
Explain the conversion between glucose-1-P and glucose-6-P.
Completely reversible reaction –> concentration determines the direction of the reaction –> Equilibrium principal.
Starting with glucose-1-P
- Phosphoglucomutase contains Ser residue bound to phosphate.
- transfers that phosphate to carbon 6 to form Glucose 1,6-bisphosphate –> phosphorylation reaction
- The same phosphoglucomutase with an -OH group now can remove cleave the phosphate from carbon 1 to form glucose-6-phosphate.
What is the main reason behind phosphorylating glucose?
The main reason to phosphorylate glucose is to prevent it from exiting the cell –> charge phosphate group doesn’t allow.
Role of liver in storage and release of glucose?
Liver stores glycogen on behalf of the whole body to keep blood glucose levels up –> once glycogen is broken down –> glc-6-P is pumped into the ER where the phosphate is removed –> hydrolysis –> Glc + Pi –> glucose can then move into the blood.
Note –> muscles store glycogen for personal use –> ATP for muscle contraction.
How is glycogen phosphorylase regulated (hormonally)?
Hormonal control
Phosphorylase b kinase –> phosphorylates both serine residues on glycogen phosphorylase –> results in the breakdown of glycogen.
PP1 –> Cleaves off the phosphate groups
In the presence of glucagon (liver) and adrenaline (muscle) –> Phosphorylase b kinase is activated
But…
Phosphorylase b kinase –> is inhibited by insulin –> both liver and muscle cells respond to insulin.
Explain the allosteric regulation of phosphorylase b.
In muscle
- Phosphorylase b is inhibited by Glc-6-P and ATP –> Both Glc-6-P and ATP are high when the muscle is at rest.
- Active when AMP concentration is high
In liver
- In the absence of hormonal stimulation –> enzyme is normally found in a form (active) –> but it is inhibited by glucose –> glucose binds and prevents enzyme activity.
Explain the regulation of glycogen synthase by phosphorylation (Hormonal)?
Glycogen synthase is a dimer with many sites of phosphorylation.
Glycogen synthase b (phosphorylated form) –> less active form.
Glycogen synthase a (not phosphorylated form) –> more active form.
Opposite to glycogen phosphorylase –> this case dephosphorylation increases activity.
Hormonal control –> Glc conc. is high –> insulin is high –> insulin inactivates glycogen synthase kinase (GSK3) –> leaving more in the active form.
Explain the allosteric control of glycogen synthase.
Allosteric –> more important
- Glucose-6-P is an allosteric activator of glycogen synthase –> Binds to the b form –> making it more active –> so higher Glucose-6-P means more glycogen is produced.
Explain the metabolic pathway of adrenaline (muscle) and glucagon (liver) with reference to their impact on glycogen/glucose.
Steps
- Activation of the receptor (G-protein coupled receptor)
- Activates adenylate cyclase –> ATP –> cAMP
- cAMP binds to a regulatory site on inactive Protein kinase A –> forms active PKA
- PKA phosphorylates inactive phosphorylase kinase to form the active form.
Pause –> Separate pathway
- Active PKA also phosphorylates glycogen synthase a to form the less active glycogen synthase b.
- Less glycogen produced
Play –> return to original pathway
- Active phosphorylase kinase phosphorylates glycogen phosphorylase b to form glycogen phosphorylase a (more active form).
- Results in more breakdown of glycogen to glucose.
Explain the impact of insulin on liver and muscle cells.
Insulin –> liver and muscle cells
- Insulin binds to the insulin receptor
- The insulin receptor is a tyrosine kinase receptor –> phosphorylates insulin receptor substrates (IRS-OH —> IRS-P).
- IRS-P –> activates a lot of different types of protein kinases.
- Inactivates kinases such as glycogen synthase kinase –> prevents the phosphorylation of glycogen synthase –> more of glycogen synthase in active form –> glycogen synthesized.
- Whereas, PP1 is activated by insulin –> more is converted to the glycogen synthase a (active form).
Impact of Protein phosphatase 1 (PP1) on the cell?
Summary of the impact of different hormones on glycogen synthesis and breakdown.
Glucagon/adrenaline
- Phosphorylation of glycogen phosphorylase –> more active —> increased glycogen breakdown.
- Phosphorylation of glycogen synthase –> less active form –> decrease glycogen synthesis
Insulin -> high blood glucose
- dephosphorylation of glycogen phosphorylase –> less active –> less breakdown of glycogen
- dephosphorylation of glycogen synthase –> more active form –> increase synthesis of glycogen.
The relationship between glycolysis and gluconeogenesis?
Glycolysis and gluconeogenesis are both the reverse of each other.
However….
There are 3 irreversible reactions in glycolysis that need to be bypassed in gluconeogenesis.
What are the steps in gluconeogenesis?
- The reactions that are labelled are the reactions that are irreversible in glycolysis so that have to be bypassed via another reaction(s).
What is the Cori cycle?
The Cori cycle is when lactate produced in muscles is used and converted to pyruvate which than then be used to make glucose.
Explain the steps in the Cori cycle.
Glycolysis (2 Pyruvate to 2 lactate) produces two ATP molecules.
Lactate is oxidized back to pyruvate in the liver, not the muscles –> ATP cost of oxidation reactions –> 6 ATP -> energetic burden is shifted from the muscle to the liver.
Explain the reactions that convert pyruvate into phosphoenolpyruvate –> first 2 reactions of gluconeogenesis.
Step 1:
Synthesis of OAA from pyruvate by carboxylation –> CO2 is fixed by pyruvate carboxylase –> this is eventually released –> no net fixation of carbon.
Note –> this reaction requires biotin as a cofactor
Step 2:
Phosphate is added and CO2 is released using PEP carboxykinase. Uses GTP rather than ATP as a source of free energy.
Explain the role of Biotin during the carbon fixation of pyruvate to form OAA.
Why is gluconeogenesis important?
Most tissues metabolize a variety of carbon sources –> brain needs glucose, R.B.C needs glucose and rapidly contracting muscle need glucose.
However….
Glycogen is not long term storage –> readily mobilized and their stores only last 1 day in a fasted state.
- We must be able to synthesize glucose from other molecules –> gluconeogenesis –> pathway is universal.
Is gluconeogenesis an energetically costly pathway?
Yes, it is energetically very costly –> uses a lot of ATP —> but it is essential.
- Uses 6 ATP for each glucose made
Gluconeogenesis
2 pyruvate + 4 ATP + 2GTP + 2 NADH + 2H+ + 4H2O —-> Glucose + 4ADP + 2 GDP + 6 pi + 2 NAD+
What are the precursors for gluconeogenesis?
- Lactate –> produced by rapidly contracting muscle
- Some amino acids –> particularly alanine –> converted to pyruvate in the first step
- Glycerol –> released from fats —> fatty acids are broken down to acetyl CoA –> can NOT be converted to glucose.
Explain the reciprocal regulation of gluconeogenesis and glycolysis.
PFK2
FBPase2
PFK2 and FBPase2 activities are on one bifunctional protein –> regulated by phosphorylation –> which is regulated by hormones.
Insulin –> inhibits gluconeogenesis
What molecules inhibit/promote glycolysis and gluconeogenesis?
What is the role of Fructose 2,6 biphosphate in the regulation of glycolysis and gluconeogenesis
Fructose 2,6 biphosphate is synthesized as a regulator of PFK-1 and FBPase1.
How to enzymes distinguish between NAD+ and NADP+ ?
NADP+ has a phosphate tag that enzymes can identify –> allows them to distinguish.
What is the pentose phosphate pathway?
Pentose phosphate pathway is a metabolic pathway parallel to glycolysis. It generates NADPH and pentoses (5-carbon sugars) as well as ribose 5-phosphate, the last one a precursor for the synthesis of nucleotides.
Explain what happens step by step in the pentose phosphate pathway.
Takes place in the cytoplasm (Glc-6-P)
2 Main products
- NADPH
- Ribose-5-phosphate
The rate of the pathway depends on the ratio of NADP+ and NADPH –> NADP+ stimulates Glc-6-P dehydrogenase (allosteric promoter).
What is the role of glutathione in cells?
- Glutathione (reduced form) (GSH) –> used by all cells as an antioxidant in the cytoplasm –> two forms reduced and oxidized form –> reduced form is used to reduce reactive oxygen species –> maintains protein SH group in reduced form.
- It is a tripeptide –> contains a gamma amide bond –> which means that one of the side chains is forming the amide bond.
How are levels of reduced glutathione maintained in the cell?
- Glutathione reductase is used to maintain levels of reduced glutathione –> reduces GSSG to GSH –> using NADPH + H+
Explain what happens during the non-oxidative phase of the pentose phosphate pathway?
No oxidation reactions –> just molecular rearangements.
If nucleotides are not needed –> convert ribulose-5-P back into intermediates of glycolysis/gluconeogenesis –> can generate glucose-6-P.
Summarize what is happening in the pentose phosphate pathway.
Explain the different fates of glucose-6-Phosphate.
Glc-6-P is a key intermediate in carbohydrate metabolism.
Explain the structure of triacylglycerols.
Triacylglycerols/triglycerides –> composed of three fatty acids bonded to glycerol via an ester linkage. The 3 fatty acids may be different.
What are three common fatty acids?
- Palmitate –> one of the most common in animals and plants –> saturated –> no double bonds.
- Stearate –> Saturated
- Oleate –> monounsaturated
Explain the process by which fatty acids are released from adipose tissue.
Adipose tissue
- Glucagon/adrenaline binds to G-protein coupled receptor
- Activated GTP/Alpha subunit activates adenylate cyclase.
- converts ATP to AMP
- AMP activates Proteins kinase A (inactive to active)
- Kinase phosphorylates triacylglycerol lipase
- Mobilization of triglycerides from fat droplets (Perilipin)
- Phosphorylated triacylglycerol lipase –> cleaves off one fatty acid to form diacylglycerol.
- The process continues with the use of other lipases –> fatty acids diffuse out of adipose tissue
- Transfer of fatty acids by serum albumin (most prevalent in the blood)
- Tissue –> Activation by reaction with CoA
- Transport into mitochondria.
Explain the structure of Coenzyme A.
Explain the reaction required to synthesize Acyl CoA
- Carboxylate ion displaces outer 2 Phosphate groups of ATP.
- Nucleophilic attack by SH group of acyl adenylate.
The process uses the equivalent of 2 ATP
Explain the conjugation of carnitine with acyl CoA.
Acyl-CoA attacks -OH group on carnitine –> releases CoA-SH.
This is important as the inner mitochondrial membrane is impermeable to acyl-CoA.
What are the first reactions of Beta-oxidation?
Initial oxidation of Beta carbons involves 4 reactions –> releases a 2 carbon fragment as acyl CoA and acetyl CoA.
Explain the Beta-oxidation of palmitate.
Step by step reactions.
- Acyl-CoA –> undergoes oxidation by FAD –> creates a double bond and forms Enoyl-CoA
- Enoyl-CoA undergoes hydration –> hydrates the double bond –> introduces H and OH –> forms 3-hydroxylacyl-CoA.
- 3-Hydroxylacyl-CoA –> undergoes oxidation by NAD+ –> turns hydroxyl into keto –> forms 3-Ketoacyl-CoA.
- 3-Ketoacyl-CoA undergoes thiolytic cleavage –> cleaves two carbons –> forms C14Acyl-CoA and acetyl-CoA (can be used in Krebs cycle)
Note –> this process repeats itself another 6 times –> a total of 7 acetyl-CoA are released
Note –> different mechanism for unsaturated fatty acids.
Explain the catabolism of propionyl-CoA (Beta-oxidation) –> odd-numbered fatty acid chains.
Note humans don’t synthesize odd-numbered fatty acid chains –> but bacteria do so humans have developed a mechanism to deal with this.
- Carboxylation reaction of propionyl-CoA to form D-methylmalonyl-CoA.
- Stereochemical reconfiguration of D-methylmalonyl -CoA using an epimerase.
- L-Methylmalonyl-CoA undergoes another Stereochemical reconfiguration using a mutase –> moves functional groups around to form succinyl-COA
- Succinyl-CoA can be used in the Krebs Cycle or used to form OAA which can then be used to form glucose.
Explain the first step of FA synthesis (committed step).
- Bicarbonate and Acetyl CoA react –> carboxylation reaction –> Acetyl CoA carboxylase –> uses biotin as a cofactor.
- Form Malonyl CoA
Explain the mechanism by which biotin acts as a cofactor in the carboxylation of acetyl CoA to form malonyl CoA.
- Biotin binds to HCO3- –> acts as a carrier of CO2
- Biotin transfers acetyl CoA to acetyl CoA.
WHat is an important carrier protein in FA synthesis?
Intermediates in FA synthesis linked to Acyl carrier protein (ACP) —> equivalent to CoA.
Once Malonyl-ACP has been created –> what is the step by step process to form FA chains?
Cycle 1 –> Elongation phase –> adding 2 carbon molecules repeatedly.
- Acetyl-ACP + Malonyl ACP –> undergo condensation reaction.
- Acetoacetyl-ACP undergoes a reduction –> NADPH+H+ electron donor.
- D-3-Hydroxybutyryl-ACP –> undergoes a dehydration reaction (OH + H removed) –> forms double bond
- Crotonyl-ACP undergoes reduction using NADPH+H+ electron donor.
- Forms Butyryl-ACP
This cycle repeats itself over and over again building up the chain –> Malonyl-ACP acts an activated donor of 2 carbons –> Malonyl-ACP is used is because it makes the reaction energetically favourable –> acetyl-ACP wouldn’t work.
Lastly –> Thioesterase cleaves off the ACP protein.
Note –> This is all catalyzed by Fatty acid synthase –> multiple active sites.
Compare oxidative degradation of FA and their synthesis.
DIfferent cofactors
- FAD/NAD+ —> degradation
- NADPH –> synthesis.
Explain the hormonal control of fatty acid synthesis.
Explain the allosteric control of fatty acid synthesis.
What are the fates of the carbon skeleton during amino acid degradation? (Linked to TCA cycle)
- After the removal of the amino group –> all 20 amino acids are broken down into just 7 molecules, that can be used to synthesize glucose or be oxidized into the TCA cycle.
- The three categories of Amino acid indicate their eventual fate –> Ketogenic A.A can only be converted to acetyl CoA and can NOT be converted to glucose. Glucogenic A.A are broken down to OAA, pyruvate, Alpha-ketoglutarate, succinyl CoA or fumarate –> this can then be used in TCA cycle of gluconeogenesis.
Explain the general process of transamination.
- With an enzyme called aminotransferase (transaminase) –> the amino group from an amino acid is transferred to an alpha-keto acid.
The alpha-keto acid is normally alpha-ketoglutarate which forms glutamate after the transfer of the amino group.
Note - This reaction is completely reversible –> operates at an equilibrium –> directly dependent on the reactants in the cell.
Write out the equations for the transamination of alanine and aspartate.
Explain the mechanism for transamination.
- Condensation reaction between aldehyde and amine group –> forms a Schiff base intermediate –> Key step
- Then the Schiff base intermediate is rearranged to either a Quinonoid intermediate or carbanion-R1.
- Hydrolysis reaction –> releases Alpha-keto acid and pyridoxamine phosphate.
- Another alpha-keto acid comes in and reacts with pyridoxamine phosphate to form an amino acid (usually glutamate) and pyridoxal phosphate
Explain the removal of the alpha-amino group from glutamate.
Glutamate Dehydrogenase reaction
Oxidative deamination
- The reaction releases free ammonia which can be used to produce urea.
- Glutamate Dehydrogenase is one of the few enzymes that use either NADP and NAD as a cofactor. In mammals, dehydrogenase is found in the mitochondrial matrix.
Explain what happens in the Urea cycle.
- Spans two compartments in the cell –> Mitochondrial matrix (M.M) and cytosol.
1. Starts in M.M where Bicarbonate and ammonia react together to form carbamoyl phosphate.
2. Carbamoyl group gets transferred on to an ornithine molecule to form citrulline.
3. Citrulline moves into the cytosol where it undergoes a condensation reaction with aspartate (between Amino on Asp and carbonyl of citrulline).
4. Arginosuccinate molecule is cleaved to release a fumarate molecule and arginine.
5. Arginine undergoes cleavage to release urea –> ornithine is once again produced to complete the cycle.
Note –> Urea contains two nitrogens –> each round of the cycle gets rid of two amine groups –> free NH4+ and from aspartic acid.
Explain the mechanism for the first step in the urea cycle (production of carbamoyl phosphate).
1 reaction of urea production –> occurs in 3 steps
Step 1: Phosphorylation of bicarbonate
Step 2: Displacement of phosphate with ammonia
Step 3: Phosphorylate the carbonate –> forms activated carbamoyl donor.
As one can see this initial reaction requires 2 ATP
How is the urea cycle linked to the TCA cycle?
The urea cycle is linked to the TCA cycle by fumarate.
Known as the Krebs bicycle
How are amino acids synthesized?
All 20 AA are synthesized from common precursors that are intermediates of the TCA cycle and glycolysis.
Note –> essential A.As must be contained in the diet as they can not be synthesized by us.
Explain the process of ketone body synthesis.
Liver Only –> In mitochondria
Explain the process of ketone body degradation.
Occurs in liver mitochondria
Using the Krebs cycle diagram explain how bacteria and plants are able to produce glucose from acetyl-CoA.
They bypass two points in the TCA cycle where carbon is released –> glyoxalate cycle
Summary of the different pathways Glucose-6-P can enter.
Summary of the key pathways pyruvate and acetyl CoA can enter.
What are the metabolic impacts of glucagon on blood glucose?
What are the metabolic impacts of insulin on blood glucose?
