Cells and Enzymes Flashcards

Question

Draw and describe the nuclear pore complex.

Answer 1

The nuclear pore complex: About 30 different proteins 500-1000 protein molecules 3000 to 4000 NPCs per mammalian cell Mass: ~ 125 million daltons

Answer 2

For small protein molecules \< 60 kDa, diffusion is sufficient for nuclear import For larger protein molecules \> 60 kDa, diffusion is not sufficient for nuclear import, and active transport is needed

Answer 3

Nuclear proteins are recognised by a nuclear import receptor. The proteins that make up this receptor are called impartins. The complex is formed by a cargo protein and a receptor. The import receptor facilitates transport of the protein across the nuclear envelope through the nuclear pores. Once this complex reaches the nuclear lumen, the cargo protein is replaced by a Ran-GTP protein. This releases the cargo protein, the cargo is delivered to the nucleus. Ran-GTP converts to Ran-GDP, meaning ya no puede bind to the import receptor, so its free. This import receptor goes back to the cytoplasm to bind to a new cargo.

Answer 4

Respiration, ATP synthesis Heat generation(in brown fat) Fatty acid metabolism Intermediary metabolism (synthesis & breakdown of biomolecules)

Answer 5

Proteins are imported from the cytoplasm into mitochondria but mitochondria has its own DNA, which makes some of its proteins inside the organelle.

Answer 6

The signal sequence is recognised by a TOM receptor complex in the outer membrane of mitochondria. The protein binds to the Tom complex, is handed over to a channel in the outer membrane, and moves into the inter membrane space. When this occurs, the protein makes contact w an inner membrane Tim23 complex and moves into the matrix. The original signal sequence is cleaved off by signal peptidases. The mature protein reaches its destination.

Answer 7

The signal sequence binds w the Tom complex receptor, enters the channel of the Tom complex, and then binds w the Tom complex on the inner membrane. Once the signal sequence reaches the matrix it is cleaved off. A section of protein exits the Tim channel laterally and enters the membrane bilayer. The rest of the protein exits the Tom complex. You now have the mature protein w a transmembrane domain anchoring it in the inner membrane.

Answer 8

The precursor protein associates w another cytosolic HSP 70 protein which binds ATP. In order for the precursor protein enter the Tom complex, HSP 70 must be released, which is done by ATP hydrolysis. Also hay mitochondrial HSP 70 which does the same thing in the matrix. The third source of free energy is membrane potential. This is when hay mas + charge in the intermembrane space than the matrix. This creates a conc and electric gradient which is used to make ATP and drives protein transport.

Answer 9

Protein synthesis, folding, glycosylation & disulfide bond formation Protein quality control Lipid synthesis Ca2+ storage Intermediary metabolism

Answer 10

Post-translational protein modifications (glycosylation, sulfation, proteolysis which is cutting proteins) Lipid synthesis Protein & lipid sorting (eg packaging into secretory granules, plasma membrane, endosomes, lysosomes)

Answer 11

Starts with a complex of mRNA and a ribosome. This complex has a signal sequence (6 to 12 amino acids, hydrophobic, N-terminal). As the signal sequence emerges from the ribosome it's recognised by an SRP which binds to it to form a complex. This pauses translation. The new complex docs onto an SRP receptor in the ER membrane. The SRP receptor attaches the complex onto the "protein translocator". The SRP and SRP receptor are then displaced + recycled, protein synthesis resumes. As the protein is made, its threaded through the translocation channel, into the lumen of the ER. The signal peptide is cleaved off.

Answer 12

Vesicles form from the donor membrane which pinch off, cargo proteins trapped in the lumen. These travel along the cytoplasm usually along microtubules, until reaching target. Hay diffusion of the vesicles into the target compartment Remember that vesicles form with the aid of specific vesicle coats.

Answer 13

Signal sequences determine protein localisation.

Answer 14

A form: right handed helix, often found in RNA and tRNA. Ha major and minor groves which have v similar sizes It is L shaped, and is a single polynucleotide chain It has regions that are self complimentary 1 complete turn= ~11 base pairs

Answer 15

Right handed helix. Most common in cells. Has major and minor groves which provides access to proteins and chemicals to read the DNA. 1 complete turn= 10 base pairs

Answer 16

Left handed helix, rarest type. Usually positioned in front of genes. Has a role in regulating gene expression Some proteins can bind to Z DNA Z DNA forms when hay repeated alternating sequences of purine and pyrimidine bases: -5’ GCGCGCGC...-5’ GTGTGTGT...

Answer 17

DNA polymerase copies DNA strand in the presence of inhibitors that arrest DNA synthesis at A, C, G or T. The DNA strands are separated by length on a polyacrylamide gel. 700-1000 base sequences read. Eg in the image below the bases from the bottom up: CGCGCAACA etc Automated methods allow rapid DNA sequencing. Used to sequence human, yeast, bacterial, viral genome. From the sequence of DNA, using triplet code you can also infer the protein sequence of the product.

Answer 18

As well as diff DNA sequences and diff helical forms, hay other higher order DNA structures. These are: Holliday Junction: made up of 2 chromosomes. Hay exchange of DNA strands between 2 helices to form a 4 stranded junction. This helps DNA repair: if 1 chromosome is damaged, cells can copy the good DNA sequence on the other chromosome via strand invasion and repair damage. Tetraplex: form at telomeres at the end of chromosomes (in white). They have G rich sequences which fold to form a 4 stranded DNA helix

Answer 19

Primary: sequence of bases, can be analysed by DNA sequencing Secondary: helical structures, can be analysed by X ray and chemistry Tertiary: DNA supercoiling, where DNA is twisted on itself. Analysed by electron microscopy Quaternary: interlocked chromosomes

Answer 20

E. coli DNA is circular and comprises 3 x 106 basepairs. Is supercoiled: the DNA ribbon itself is twisted in space. E coli has 50 super coiled domains Supercoiling is caused by DNA gyrase enzyme. Its sister enzyme topo IV catalyses DNA relaxation

Answer 21

The super coiled DNA has energy stored in it due to unwinding and resealing the helix to get the supercoil. Therefore DNA supercoiling can facilitate the unwinding of the helix for DNA replication. Other important supercoiling feature: it reduces the size of the DNA so that the DNA will fit inside the cell.

Answer 22

Novobiocin competitively inhibits the binding of ATP to gyrase. Fluoroquinolones inhibit DNA resealing by gyrase/topo IV, so acts as an antibiotic

Answer 23

3bn basepairs of DNA per cell, organised into 23 pairs of linear chromosomes. DNA is complexed w histones to form a nucleoprotein complex called chromatin. Nucleosome is a basic building block of chromatin. It has a core of 8 histones- 2 copies of histones 2A, 2B, 3 and 4. Nucleosome is seen as beads on a string. Histones have + tails which interact with - charged phosphate groups on DNA. This allows DNA to wind around histones. DNA gets compacted by a factor of 6

Answer 24

This refers to the carbon atoms of the sugar to which the oxygens on the phosphate are linked.

Answer 25

DNA is chemically unstable. Damage must be sensed and continually repaired to maintain DNA's cellular functions.

Answer 26

Spontaneous: eg loss of bases, or hydrolysis of C to U Chemicals and radicals generated by oxidative metabolism change base structure and insert between bases. (Cyclophosphamide, and intercalators like doxorubicin are widely used as anticancer drugs in this way) Radiation: UV produces thymine dimers. Ionising radiation (X-rays, gamma rays) break DNA chromosomes to cause leukaemia

Answer 27

DNA repair maintains genome stability. Patients with xeroderma pigmentosum are v prone to skin cancer. This is bc tienen a defect in excision repair that deals w UV damage to DNA. Other cancer prone families have DNA repair defects, predisposing them to eg breast/colon cancer

Answer 28

DNA replication is initiated at specific sites on DNA called replication origins Replication origins are recognised by an initiation complex DNA at the origin unwinds to form a replication bubble which allows access to DNA polymerase synthesis occurs in phase (S) of the cell cycle and involves complete unwinding of the parental DNA

Answer 29

G1: Protein synthesis occurs. Production of new organelles, rapid growth. S: DNA replication G2: Cell growth, some organelles divide. Hay a buildup of energy reserves. Respiration occurs in G2 and G1 Bc bacteria have less DNA, most of the bacterial cell cycle is S phase

Answer 30

Hay 2 replication forks, where the parental DNA duplex unwinds to make the separated strands. This means that replication is bi directional in bacteria

Answer 31

In eukaryotic cells, DNA replication initiates at multiple origins, eg here hay 4 replication origins where the parental strands unwind. Overtime the parental strands move away on either side of the replication bubble. Replication bubbles get bigger until they fuse. This ensures eukaryotic replication is efficient

Answer 32

Translesion polymerases are involved in allowing replication forks to bypass irreparable damage Gamma polymerases= specific to mitochondrial DNA

Answer 33

Acts in 5′ to 3′ direction. Utilises A-T and C-G base pairing to synthesise new DNA strand. Requires a DNA template (parental strand), a DNA or RNA primer, the four deoxyribonucleoside triphosphate (dNTP) building blocks and Mg2+ ions. Has a proof-reading editing function

Answer 34

DNA polymerase adds on to the 3’ OH end of the primer to synthesise new DNA 3’ OH from previous base eliminates the phosphate, thus releasing pyrophosphate. A new phosphate group forms in the centre to create a new nucleotide Polymerase adds a nucleotide to the 3'-OH end, extending the chain in the 5′ to 3′ direction

Answer 35

On the leading strand we add nucleotides to the 3' end of the growing strand which travels in the same direction as the replication fork. Lagging strand: adding nucleotides to the 3' end means this travels in the opp direction of the replication fork. So to replicate the newly exposed template strand from the fork, keep reinstating DNA synthesis on this strand. The DNA on the lagging strand is made up of Okazaki fragments, which have to be linked juntos to make intact DNA.

Answer 36

Primase: links a small piece of RNA to the DNA. Pol alpha adds a small amount of DNA onto the RNA. This is then used by pol Delta to make more DNA. DNA ligase: joins the Okazaki fragments DNA binding proteins protect DNA from degradation Helicase breaks the H bonds between bases during replication Topoisomerase removes intertwining every 20 base pairs. This is bc intertwining may repress DNA synthesis. Repair DNA polymerase???????

Answer 37

Replication error rate is v low- errors during synthesis form kinks which are detected by the DNA polymerase and mismatch repair system The base pairing and proof reading/editing function of the enzyme-mismatch repair system corrects most polymerase errors Inherited defects in mismatch repair genes are involved in cancer

Answer 38

Clinically important drugs inhibit DNA synthesis

Answer 39

Enzymes are proteins that catalyse chemical reactions. They ⇡ rr by up to 10bn fold!. They show specificity, don't alter reaction eqm and are unchanged at end of reaction Their various functions inc: digestion, fibrin clotting catalysed by thrombin defence: immune system, activation of complement movement: muscle actomyosin is an ATPase nerve conduction: membrane ion pumps for Na+, Ca2+

Answer 40

nuclease: break down nucleic acids polymerase: put things together kinase: transfer phosphate groups

Answer 41

Enzyme defects can cause diseases: phenylketonuria: children cannot convert Phe to Tyr, which is toxic for the brain. Treatment= low Phe diet Glycogen storage disease: can't mobilise glycogen in liver, so it's difficult to maintain blood glucose levels Tay-Sachs disease: defect in processing a membrane ganglioside- neuronal damage and death

Answer 42

Antibiotics block enzymes for cell wall synthesis eg penicillin Anti-inflammatory drugs eg aspirin inhibit prostaglandin, a part of the inflammatory response Anti cancer drugs: methotrexate is a folic acid analog-it acts as a competitive inhibitor to enzymes that use folic acid. Folic acid is converted to a co enzyme to make bases for DNA synthesis-competitive inhibition by methotrexate reduces base production.

Answer 43

Facilitate reaction by decreasing the free energy of activation of the reaction(lowers activation energy) free energy of activation needs to be negative for a reaction to be feasible-the energy level of the product should be less than the substrate this free activation energy needs to be reduced by enzymes so the reaction can happen in the body

Answer 44

Enzymes have active sites. These are 3D cavities that bind substrate(s) using electrostatic, hydrophobic, H bonding and VDW interactions When a substrate binds to the enzyme, the binding energy is enough to distort and break substrate bonds. This reduces free energy of activation

Answer 45

X-ray crystallography: purify a large amount of protein, crystallise it and shoot a v powerful x-ray beam towards it. Molecules in the crystal diffract the x-ray to leave a diffraction pattern. This helps work out the structure of individual molecules in the crystal.

Answer 46

Lock and key: substrate shape is a direct complement to the shape of the a.s. Induced fit: as the substrate binds, enzyme begins to change shape to become complementary to the shape of the substrate. Most enzymes work this way

Answer 47

Enzymes bring substrates v close to each other and bring molecules juntos at the a.s. This= approximation The binding energy constrains the movement of the substrate, positioning it so they can react optimally. This lowers free activation energy

Answer 48

Enzymes stabilise + and - charges in the transition state bc the enzyme has residues that neutralise these charges Enzymes close over the substrate and eliminates water. Making the reaction hydrophobic increases rr Enzymes use co-factors to bring a different chemistry to the reaction, this provides a different route which lowers free ae.

Answer 49

Sometimes bonds in the substrate must be broken to make the product. The enzyme uses binding energy to strain bonds in the e-s complex so it's easier to break in the transition state. You thus need less free energy to reach the transition state. eg lysozyme recognises polysacs on bacterial cell walls. it cleaves the **β**1-4 linkage, straining the bond in the cw. Water can now enter and cause lysis

Answer 50

Is proof for existence of active sites. Rate against [S] is plotted. Km is the Michaelis constant, which varies significantly for diff enzymes. It tells the binding affinity of the enzyme: lower the Km, tighter the binding of the substrate Vmax is the max possible velocity when all a.s are full. The graph levels off at higher substrate levels, showing a.s are a limiting factor Vmax/[Enzyme] total gives the Kcat number. This= turnover rate of an enzyme when working at full force

Answer 51

Competitive inhibition: this is when an enzyme and inhibitor both bind to the active site Vmax is unaltered at infinite substrate levels: the inhibitor is always outcompeted so Vmax stays same Km increases: it's harder for the substrate to attach as it must outcompete the inhibitor for the a.s. To get to half of max velocity, you need much more substrate to outcompete the inhibitor.

Answer 52

Non competitive inhibition: when the inhibitor binds to a site other than the a.s Vmax decreases-the inhibitor works through the enzyme to change the reaction rate Km is unaltered bc no hay competition for the a.s. Substrate still has the same affinity to the enzyme

Answer 53

If unregulated, enzymes will run out of its energy sources very quickly. Regulation mechanisms: Controlling gene expression of enzyme controls enzyme, eg lac operon: presence of lactose removes inhibitor which allows production of enzyme Compartmentalisation: enzymes are targeted to dependent organelles. They're sorted by barcode on their p.p chain which instructs where to go Covalent modification changes enzyme shape/activity. Eg phosphorylation attaches a -2 charge phosphate. This changes enzyme folding and thus a.site shape. Feedback inhibition: as the product builds up, the enzyme begins to get inhibited

Answer 54

Allosteric regulation: a regulatory molecule (acting at a pocket distinct from the a.s) changes the a.s conformation to decrease enzyme activity. This controls the flux of material through a metabolic pathway. eg CTP binds to allosteric inhibitor sites on the regulatory subunits. It changes the shape to bring the 2 hemispheres juntos. the substrates cannot get to the active site inside.