Biochemistry Flashcards
What are the two cellular pathways involved in metabolism?
Catabolism
Anabolism
Define metabolism
The set of life-sustaining chemical interactions that occur within an organism’s cells
What is a carbohydrate?
Carbohydrates are organic molecules consisting of carbon, hydrogen and oxygen
What is a monosaccharide?
Simplest unit as glucose, cannot be further hydrolysed to simpler compounds
What is a disaccharide?
Two monosaccharides are joined by a glycolic chain
I.e.: sucrose= glucose+ fructose
What is an Oligosaccharide?
3 to 10 monosaccharides joined together
What is a Polysaccharide?
More than 10 monosaccharides joined together
How many ATP molecules do normal cells produce during respiration (aerobic+anaerobic)?
2 ATP from anaerobi
38 ATP from aerobic
How many ATP cell do cancer cells produce during respiration?
2 ATP
What is the effect of cancer on cells during respiration?
Cancer cells are more glycolysis-dependent , therefore they require more sugar
How can we target for cancel cell therapy?
Through “fake glucose”(2 DG)= cannot go through glycolysis
if cancer cells are more glycolytic-dependent, then they require sugar more; this means that a treatment which gives them “fake glucose” (which cannot go through glycolysis) will more adversely affect cancer cells rather than healthy cells
What is the major sugar in our diet?
Glucose
All cells in our body can use glucose as an energy course
True or false?
True
What happens to glucose once at the intestine?
Glucose leavens the intestine via the hepatic portal vein and arrives first at thrived
Hepatocytes take up glucose where it is converted to pyruvate in the cytoplasm
What is Glycolysis?
Conversion of glucose to pyruvate in the cytoplasm
What are the phases of Glycolysis?
The energy investment phase
The energy generation phase
What are the steps in the Energy Investment Phase?
- Conversion of glucose to glucose-6-phosphate
- Conversion of glucose-6-phosphate to fructose-6-phosphate
- Conversion of fructose-6-phosphate to fructose-1,6-bisphosphate
- Conversion fructose-1,6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate
- Conversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate
What are the steps in the Energy Generation Phase?
- Conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphate
- Conversion of 1,3-bisphosphate to 3-phosphoglycerate
- Conversion of 3-phosphoglycerate to 2-phosphoglycerate
- Conversion of 2-phosphoglycerate to phosphoenolpyruvate
- Conversion of phosphoenolpyruvate to pyruvate
What I X-ray crystallography?
The protein analysis technique that can be performed to generate quaternary structures and see protein structures
Clinically, why do you think it is useful to know protein crystal structures?
Clinically speaking, it is important to know protein structures as it allows you to identify which abnormalities may be responsible for particular pathologies, and allows for the application of research methods such as drug
What are the stages of Cellular respiration?
Stage 1: Generation of Acetyl CoA
Stage 2: Citric Acid Cycle
Stage 3: Oxidative Phosphorylation
What happens during the generation of Acetyl coA
Pyruvate oxidation first involves its movement across the mitochondrial membrane by the pyruvate carrier
A cell can use different fuels other than glucose (via glycolysis) to generate acetyl-CoA. It can also use amino acids and fatty acids
True or false? So what does this mean?
True
So carbohydrates, fat and protein all feed into the citric acid cycle
What happens in lipid metabolism?
- Minimal digestion by lipases in saliva
- Stomach stores and emulsifies fatty food. Gradually transferred to duodenum.
- Small intestine: Lipases break fat into free fatty acids that are absorbed by the gut epithelial cells where they are resynthesized into triglycerols
What are Bile salts?
detergent substances- emulsify fat,
allowing access of enzymes to digest fat.
What are lipoproteins?
Lipids complex with protein to become soluble aggregates
What are the products of lipid digestion?
Glycerol
Free fatty acids
Monoacylglycerols
Diacylglycerols
What are saturated fatty acids?
Simplest form of lipid, usually part of more complex lipid molecules.
All carbons of the tail are saturated with hydrogen atoms (no C=C bonds)
I,e. Stearic acid
What are unsaturated fatty acids?
Not all carbon atoms saturated with hydrogen atoms. Some C=C bonds.
.
I.e.: linoleum acid
What are the major components of dietary fats?
Trialcylglycerol /triglyceride & cholesterol
What are triglycerides ?
Fatty acids stored as an energy reserve (fats and oils) through an ester linkage to glycerol to form triglycerides/triacylglycerols
What happens during lipid digestion?
Lipases in the saliva and in the small intestine break triacylglycerol into glycerol and 3 free fatty acids.
Short chain fatty acids are absorbed directly into the blood stream from the gut epithelial cells and transported to the liver
Longer chain fatty acids and those still attached to glycerol (monoglycerides) are taken up into the ER of the gut epithelial cells and converted back to triacylglycerol for transportation.
What happens to short chain fatty acids after triacylglycerol has been broken down by lipase?
Short chain fatty acids are absorbed directly into the blood stream from the gut epithelial cells and transported to the liver attached to albumin
What happens to longer chain fatty acids after the breakdown of triacylglycerol by lipase?
Longer chain fatty acids and those still attached to glycerol (monoglycerides) are taken up into the ER of the gut epithelial cells and converted back to triacylglycerol for transportation.
What lipoproteins are present in the gut?
Chylomicron VLDL: very low density lipoprotein IDL: intermediate density lipoprotein LDL: low density lipoprotein HDLm high density lipoprotein
What is the role of LDL in lipid transport?
LDL transports cholesterol, phospholipids, triglycerides (bad cholesterol) from liver to peripheral tissues including adrenal glands and gonads
What is the role of HDL in lipid transport?
The role of HDL is to collect fat molecules such as phospholipids, cholesterol, and triglycerides in the cells of the body and transport it to the liver to be broken down
What is another name for HDL and why?
HDL is sometimes known as “good” cholesterol because high concentration of this lipoprotein usually correspond to healthier blood vessels and lower risk of atherosclerosis
What is the composition of HDL?
40-55% protein
3-10 % Triacylglycerol
15-20% cholesterol/CE
What is LDL composed of?
20-25 % protein
7-15% Triacylglycerol
45-50 % cholesterol/ CE
What is another name of LDL and why?
It is sometimes known as “bad” cholesterol because elevated concentrations of LDL are an indicator of an underlying disease such as atherosclerosis and other cardiac diseases.
What is the composition of IDL?
10-20 % protein
20-30 % Triacylglycerol
40-45 % cholesterol/CE
When IDL not present in the blood?
When fasting
What is the role of VLDL in lipid transport?
This lipoprotein is responsible for the transportation of synthesized triglyceride from the liver to the adipose tissue for storage
What is the composition of VLDL?
5-10 % protein
50-65% Triacylglycerol
4-8 % cholesterol/CE
What is the composition of a Chylomicron?
1-2% protein
85-90 % Triacylglycerol
4-8 % cholesterol/CE
What is the role of chylomicrons in lipid transport?
These lipoproteins are responsible for the transportation of triglycerides in the gastrointestinal tract to other parts of the body, such as the liver, skeletal tissue, and adipose tissue.
What is the major source of energy of cardiac/skeletal muscle?
Fatty acids released from Triacylglycerol
What happens during LDL transport of lipids ?
Fatty acids Andre broken down to Acetyl-Co A (via b-oxidation) and glycerol can vibe recycled to glucose via Gluconeogenesis (in liver)
Triacylglycerol are stored in adipocytes until it is needed
How is Acetyl co A produced from Fatty acids?
Via B-oxidation pathway to produce energy
What happens during B-oxidation of fatty acids?
1- Transport of long chain fatty acids into mitochondria
2- Fatty acids primarily enter a cell via fatty acid protein transporters on the cell surface.
Fatty acid transporters include fatty acid translocase (FAT/CD36), tissue specific fatty acid transport proteins (FATP), and plasma membrane bound fatty acid binding protein (FABPpm)
3- Once inside the cell, a CoA group is added to the fatty acid by fatty acyl-CoA synthase (FACS), forming long-chain acyl-CoA.
4- Carnitine palmitoyltransferase 1 (CPT1) converts the long-chain acyl-CoA to long-chain acylcarnitine to allow the fatty acid moiety to be transported across the inner mitochondrial membrane via carnitine translocase (CAT), which exchanges long-chain acylcarnitines for carnitine.
5- CPT2 then converts the long-chain acylcarnitine back to long-chain acyl-CoA. The long-chain acyl-CoA can then enter the fatty acid β-oxidation pathway, resulting in the production of one acetyl-CoA from each cycle of β-oxidation
6- This acetyl-CoA then enters the TCA cycle( tricarboxylic acid cycle) . The NADH and FADH2 produced by both β-oxidation and the TCA cycle are used by the electron transport chain to produce ATP.
How many ATP does each FADH2 provide?
2 ATP
How many ATP molecules does each NADH provide ?
3 ATP
What happens in the TCA cycle ?
Acetyl CoA is converted to CO2 and H20
How many molecules of ATP does each Acetyl CoA molecule provide?
12 ATP
What happens when B-OXIDATION OF FATTY ACIDS WITH AN ODD NUMBER OF CARBONS occurs?
produces an intermediate with three carbons called propionyl-CoA, which cannot be oxidized further in the beta-oxidation pathway.
What are the steps in B-oxidation of odd-chain fatty acids?
(1) carboxylation – absolute requirement of biotin as co-enzyme
(2) Conversion of D-isomer to L-isomer
(3) Synthesis of succinyl CoA
(4) Succinyl CoA enters citric acid cycle directly
What are the steps of fatty acid B-oxidation?
Oxidation : to create FADH2 + fatty acyl group with a double bond in the trans configuration
Hydration: to put a hydroxyl group on carbon 3 in the L configuration
Oxidation: of the hydro group to make a ketone
And NADH
Thiolytic cleavage : to release Acetyl-CoA and a fatty acid two carbons shorter than the starting point
When will fatty acids B oxidation stop?
It will proceed until only 2 carbon remain
What are the steps of energy metabolism/ cellular respiration in normal cells?
Stage 1: Glycolysis
Stage 2: Krebs cycle (citric acid cycle)
Stage 3:Electron transport chain
What are the steps of energy metabolism/ cellular respiration in cancer cells?
Glycolysis ONLY
What is stage 2 of cellular respiration?
What happens ?
Citric acid cycle
The oxidation of the 2 carbons in acetyl-CoA in the citric acid cycle to form 1 x GTP/ATP, 2 x CO2 and 4 pairs of electrons
What are the steps of the Citric acid cycle?
Step 1: Synthesis of citrate from acetyl coenzyme A (acetyl CoA) and oxaloacetate.
This first step of the citric acid cycle is an important regulatory point.
Step 2: Isomerisation of citrate to isocitrate by aconitase.
Step 3: Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase. The reaction reduces NAD+ to form the reduced electron carrier NADH. A carbon atom is also removed as CO2.
Step 4: Oxidative decarboxylation of α-ketoglutarate to succinyl CoA by the α-ketoglutarate dehydrogenase complex. The reaction reduces NAD+ to form the reduced electron carrier NADH – this is subsequently used in the electron transport process of cellular respiration. A carbon atom is also removed as CO2.
Step 5: Cleavage of succinyl CoA to form succinate catalysed by succinate thiokinase (also called succinyl-CoA synthetase).
Step 6: Oxidation of succinate to fumarate by succinate dehydrogenase. The reaction reduces FAD to form the reduced electron carrier FADH2 used in the electron transport process
Step 7: Conversion of fumarate to malate is carried out by fumarate hydratase and requires the addition of H2O
Step 8: The oxidation of malate to oxaloacetate by malate dehydrogenase. The reaction reduces NAD+ to form the reduced electron carrier NADH used in the electron transport process
What regulates citrate synthase (step 1 citric acid cycle)?
In humans it is inhibited by its product (citrate) and regulated by the availability of its substrates.
What is the role of citrate?
Citrate is an inhibitor of the glycolysis enzyme phosphofructokinase-1 – the rate limiting enzyme of glycolysis.
What is Isocritate (citric acid cycle step 3)?
Isocitrate is a chiral secondary alcohol which can be more easily oxidised than citrate (which is a tertiary alcohol and cannot be oxidised without breaking a strong carbon-carbon bond).
What is α-ketoglutarate dehydrogenase complex (citric acid cycle step 4)?
This enzyme complex is an aggregate of multiple copies of three enzymes with cofactors These cofactors are Thiamine Pyrophosphate, lipoic acid, FAD, NAD+ and CoA.
Step 5 of the citric acid cycle is coupled with what?
This reaction is coupled with the phosphorylation of either GDP to GTP or ADP to ATP. In tissues that are dependent on oxidative metabolism (such as brain, heart and skeletal muscle) succinate thiokinase is usually coupled with ADP/ATP. In biosynthetic / anabolic tissues (such as liver, kidneys) succinate thiokinase is coupled with GDP/GTP.
How many times is FAD pH2 formed in the citric acid cycle?
Once
How many times is NADH formed in the citric acid cycle?
3 times
The 2 carbon atoms from the Acetyl CoA are released from the cycle as?
Carbon dioxide
The 2 carbon atoms from the Acetyl CoA are released from the cycle as ?
Carbon dioxide
How much energy (ATP) is produced by the citric acid cycle?
Oxidation of NADH in the electron transport chain produces 3 ATP molecules.
Oxidation of FADH2 produces 2 ATP.
In total the citric acid cycle produces 12 ATP molecules for each molecule of acetyl CoA.
Ehm does decarboxylation occurs in the citric acid cycle?
Twice:
Step 3 and step 4
What a Hellenes in stage 3 of cellular respiration?
Electron transport chain
During this the reduced electron carriers get re-oxidised and ATP is synthesised. This process is catalysed by membrane bound enzymes in the inner mitochondrial membrane. The inner membrane is extensively stacked and folded into projections called cristae which greatly expand its surface area.
How many membrane does the mitochondria have?
2
Inner membrane: impermeable to protons. Only select molecules can pass through the inner membrane via carrier or shuttle proteins (e.g. pyruvate carrier protein)
Outer membrane: permeable to small molecules
What is the respiratory chain?
The respiratory chain is an efficient process for producing ATP from both FADH2 and NADH and consists of 5 complexes. – These are all located in the inner mitochondrial membrane.
How many complexes are involved in the respiratory chain in the electron transport chain and where are they found?
5
Inner membrane of mitochondria
Some complexes contain cytochromes, what is their function?
Transport electrons down the electron transport chain
What happens to the electrons in the electron transport chain?
They are converted into water by the reduction of oxygen by complex IV
What do Complexes I, III and IV do?
Receive electrons + pump protons into intermembrane space using energy gained by the transport of the electrons
What do protons do in the intermembrane space?
Form an electrical gradient between the intermembrane space and the mitochondrial matrix
What is Complex V?
Complex V is the enzyme ATP synthase which synthesises ATP (from ADP and inorganic phosphate) using the proton gradient.
The protons pass through its proton channel driving the catalytic process of this complex.
What are the steps of the electron transport chain?
Step 1- Some of the complexes contain cytochromes that transport electrons down the electron transport chain. Coenzyme Q (ubiquitone) and cytochrome c also participate in the electron transport.
Step 2- Complexes I, III and IV not only receive electrons but also pump protons into the intermembrane space. This is done using the energy gained by the transport of the electrons.
Step 3- The protons form an electrical gradient between the intermembrane space and the mitochondrial matrix.
Step 4- Complex V is the enzyme ATP synthase which synthesises ATP (from ADP and inorganic phosphate) using the proton gradient.
Step 5- The protons pass through its proton channel driving the catalytic process of this complex.
Step 6- The electrons that travel down the electron transport chain are supplied by the reduced electron carriers NADH and FADH2.
Step 7- Complex II is the enzyme succinate dehydrogenase of the citric acid cycle, that catalyses the oxidation of succinate to fumarate. This produces the reduced electron carrier cofactor FADH2
from FAD. FADH2 transfers its electrons to coenzyme Q.
Step 8- The electrons that travel down the electron transport chain are supplied by the reduced electron carriers NADH and FADH2.
Step 9- coenzyme q then transfers the electrons to complex III
Step 10 - The transport of electrons again produces the energy for complex III and IV to pump more protons into the inner mitochondrial space. At complex IV the electrons and the free protons are finally brought together with oxygen form water.
What is Coenzyme II in the electron transport chain?
Is the enzyme succinate dehydrogenase of the citric acid cycle, that catalyses the oxidation of succinate to fumarate.
This produces the reduced electron carrier cofactor FADH2 from FAD.
What is Coenzyme Q?
Coenzyme Q is not a protein it is a lipid-soluble quinone.
It is called ‘ubiquitinone’ because it is ubiquitous in biological systems.
It can accept electrons from complex I, complex II and other mitochondrial dehydrogenases
What is the secondary energy storage? Where is it stored?
Excess glucose is stored as glycogen (secondary energy store)
- Glycogen is stored mainly in the liver where it is a source of glucose for all other organs.
- Glycogen synthesis requires energy
Where does fatty acid synthesis occurs?
In the cytosol
What are fatty acids synthesised from?
Acetyl Co A
Palmitate (C16) primarily produced as an end product
The production of acetyl CoA in the matrix of the mitochondrion is the first stage of what?
Fatty acid synthesis
What are the steps for fatty acid biosynthesis?
Step 1- production of cytosolic Acetyl CoA
Step 2- carboxylation of Acetyl Co A to malonyl Co A - rate limiting step of fatty acid biosynthesis
Step 3-
What happens during step 2 of fatty acid biosynthesis: carboxylation of Acetyl Co A to malonyl Co A?
- An acetyl group is transferred from acetyl CoA to Fatty Acid Synthase (its ACP domain)
- The 2 carbon fragment is transferred to another, temporary holding site on the FAS enzyme (the thiol group of a cysteine residue)
- The now vacant ACP domain accepts a 3 carbon malonyl group from malonyl CoA
How is Fatty acid stored as Triacylglycerol (TAGs)?
(1) The fatty acids are usually not the same type within a triacyglycerol molecule
(2) Glycerol 3-phosphate (to form glycerol) is produced by liver and adipocytes
(3) TAG only slightly water soluble – stored as oily droplets in WHITE adipocytes
(4) TAG stored in brown adipocytes serve as a source of heat through non-shivering thermogenesis.
(5) Liver exports TAGs packaged with other lipid and apolipoproteins to form very low density lipoproteins (VLDLs)
Why is fat an energy reserve?
More energy release than carbohydrates
Denser than carbohydrates
Insolubility of fat does not affect intracellular osmotic pressure.
Most of the energy in fat released as OXIDATION of FATTY ACIDS for many tissues (except brain)
What is Kypolysis and how is it achieved?
Release of fatty acids from Triacylglycerols
Achieved by lipase:
(1) adipose triglyceride lipase
(2) hormone-sensitive lipase
(3) monoacylglycerol lipase
What lipase are involved in lipolysis?
(1) adipose triglyceride lipase
(2) hormone-sensitive lipase
(3) monoacylglycerol lipase
What happens during Lipolysis?
Fate of glycerol: transported through blood to liver, phosphorylated to glycerol-3-phosphate to form new TAGs
Fate of fatty acids: bind to albumin, transported to tissues / cells, activated into CoA derivatives, oxidised in mitochondria.
Case study
A 34-year-old African-American man presented with fever and shortness of breath. Shortly afterwards, he developed pancreatitis and was treated with clindamycin (an antibiotic) and primaquine (an anti-malarial).
- Four days onto therapy haematuria was noted
- The patient’s haemoglobin fell from 11g/dL to 7.4g/dL, total bilirubin increased from 1.2 mg/dL to 4.3 mg/dL, and lactate dehydrogenase (LDH) increased from 248 IU/L to 612 IU/L.
The patient is most likely suffering from glucose-6-phosphate dehydrogenase (G6PD) deficiency
- G6PD is an enzyme involved in the pentose-phosphate pathway that is important for redox metabolism for our cells. It is especially true in erythrocytes, as it is the only source of NADPH.
- G6PD maintains NADPH levels, which in turn protects cells against oxidative stress.
- The haemolysis is due to primaquine, as it is an oxidant drug.
- A contraindication for primaquine is G6PD deficiency, as it has a risk of haemolytic anaemia – which we have seen here with our patien
What are the techniques used to analyse gene or protein?
PCR polymerase chain reaction
Protein analysis
What is PCR polymerase chain reaction?
Consists of repeated cycles of
denaturation - breaking apart DNA strands at 94-95 degrees
Annealing- primer binding to DNA at 68 degrees
Elongation- DNA polymerase synthesises DNA at 72 degrees
What are primers?
Custom DNA sequences that bind to a DNA sequence of interest through complimentarity
DNA sequence is GAACTA , the primer could be CTTGAT
Wha is protein analysis ?
Native and post-translational modified- is important for determination of biological function
Two techniques:
Western blotting : semi-quantitative but high sensitivity
ELISA : quantitative
What can be used to detect HIV?
ELISA test
Western blotting
