Cells and Enzymes Flashcards
State the definition for a cell.
Cells are the fundamental unit of all living things on earth. They take nutrients and free energy from their surroundings and make copies of themselves.
Cells normally range between 1– 100µm in diameter.
What are universal features of all cells on Earth?
1) All cells have a plasma membrane, usually a phospholipid bilayer.
2) All cells exchange molecules w surroundings
3) Cells communicate w receptor, intracellular signalling and effector proteins.
4) All cells store hereditary genetic info as DNA
5) Protein synthesis: Cells translate RNA into protein in the same way
6) Cells have universal organelles, compartmentalisation.
7) Life as a ‘pattern in flux’: molecules are constantly being replaced.
8) Evolution from common ancestor, grouped into 3 domains
Describe the three levels of homeostasis.
Chemical: molecules in cells or blood (biochemistry)
Cellular: maintenance of subcellular structures; proper distribution of organelle-specific proteins, lipids etc (cell biology)
Systemic: BP, water balance, food intake, body temp and energy storage etc (physiology)
Describe and explain the structure of DNA
DNA is 2 antiparallel polynucleotide chains coiled into a double helix, held by H bonds between AT and CG pairs. ACGT is the coding element of the DNA.
Each nucleotide has a nitrogenous base, phosphate group and deoxyribose sugar. Phosphodiester bonds hold the phosphate and sugar backbone.
Phosphate bonded to 3C on one side, and 5C on the other to form a 3, 5 phosphodiester linkage. DNA structure runs 5’ to 3’
Phosphate and deoxyribose sugar are constant, meaning they dont change. One complete turn= 10 base pairs
Complementarity of DNA strands helps repair and replication
In the structure of DNA, what are the 2 types of nitrogenous bases?
Purines and Pyrimidines.
Purines= 2 nitrogen containing rings. A and G are purines.
Pyrimidines= 1 nitrogen containing ring, T and C.
The bases pair by H bonding, purine to pyrimidine.
CG bond is stronger as it makes 3 H bonds (bond length 1.08nm). AT bond is weaker as it makes 2 H bonds (bond length 1.11nm)
Draw the basic structure of an amino acid, followed by its structure at different pH levels
Name the four principal groups of amino acid side chains
Amino acids can be polar or nonpolar.
Within polar amino acids, they can be + charged, - charged or uncharged
draw the formation of a peptide bond by condensation.
The left side is the amino terminal end and the right side is the carboxyl terminal end
The sequence of aa.s= primary structure of a protein
What are Beta pleated sheets?
Beta pleated sheets: Part of the secondary structure. Different parts of a protein oreintate themself in parallel or antiparallel. This is stabilised by H bonds entre adj strands
Parallel means that the N and C groups are the same on one side
Antiparallel means the N and C groups alternate (see image)
The side chains (see purple) of the a.a point to opp sides of the sheet.
Fibroin is a key example
What is alpha helix?
A secondary structure
H Bonds are formed between peptide bonds within a single strand of protein. Side chains protrude from sides of the helix. Sometimes residues are hydrophilic on one side of the helix and hydrophobic on the other.
Describe the tertiary structure
Helices and sheets are folded up into more densely packed globular structures.
The structures are stabilized by cv disulfide bonds entre cysteine residues.
Some enzymes need additional chemical components, or cofactors to form the completed holoenzyme. A holoenzyme contains both the pp chain and cofactor.
Apoenzyme + Cofactor —> Holoenzyme
What is the Quaternary structure?
Haemoglobin has 4 polypeptides + 4 heme groups
When 2 pp chains are bonded juntos. The same bonds in tertiary structure hold the subunits juntos.
describe the noncovalent interactions in proteins.
Non-covalent Interactions in Proteins:
H Bonds between neutral groups and peptide groups.
Ionic attraction between + and - side chains. Ionic repulsion between like charges on different chains.
Hydrophobic interactions bring hydophobic side chains into close proximity to push them away from aqueous surfaces
VDWs forces occur between any atoms close to each other
Can proteins form covalent interactions?
Proteins can also form covalent interactions to stabilise tertiary and quaternary structures. This is formed between Cys residues, where an oxidation reaction forms a disulphide bond.
This may crosslink a single pp chain (3° structure) or 2 separate molecules (4° Structure)
Describe and explain the shape of various protein types.
Water soluble proteins are often globular: hydrophilic residues mostly on the outer surface, hydrophobic residues in the protein. May assemble into filaments (actin) or tubes (e.g. tubulin).
In membrane proteins, hay externally located hydrophobic residues that interact w the membrane lipids. May have hydrophilic central channels.
Fibrous proteins like collagen have a triple helix (NOT alpha helix). Collagen molecules may assemble into long fibres or sheets. Other fibrous proteins=myosin and keratin.
describe the mutation leads to sickle cell anaemia.
Sickle-cell anaemia is caused by a point mutation on a beta Hb chain: Glu (negative) is replaced by Val (hydrophobic ) at position 6.
In the deoxygenated state, the mutant (HbS) molecules stick juntos and form insoluble fibres.
Precipitation of the major protein in the cell distorts the normal disc shape to the characteristic “sickle shape” and blocks capillaries. The fragile cells break⇢ anaemia.
Explain DNA replication
It is making more DNA.
DNA is unzipped.
Strands separate and each strand acts as a template.
Free nucleotides line up against comp base pairs.
DNA polymerase joins new nucleotides together.
DNA ligase joins DNA to form two identical double strands of DNA.
Each one consists of one new strand and one original strand.
This is semi conservative replication
What is the difference between DNA and RNA?
RNA is: single-stranded, has ribose instead of deoxyribose and U instead of T. Hay diff types of RNA in mammalian cells. Only mRNA encodes proteins
Outline the roles of mRNA, rRNA and tRNA
mRNA: codes for proteins
rRNA: form the basic structure of the ribosome and catalyse protein synthesis
tRNA: central to ps as adaptors between mRNA and aa.
Give the stop and start codons
Start: AUG
Stop: UAA, UAG UGA,
Also hay 3 reading frames in mRNA
Each frame translates a completely different peptide. Only 1 frame is used for each mRNA, determined by AUG start codon position.
Describe tRNA molecules
tRNA is attached to aa.s in a cv bond. The anti codon makes contact with the mRNA by recognising its triplet codon via comp base pairing.
Which antibiotics affect ps?
eg. tet binds to 30S and prevents tRNA binding
Chloramphenicol binds to 50S and blocks peptide bond formation
Limitations of Antibiotics Acting on Protein Synthesis?
- No action on viruses, which use the host’s protein synthesis machinery
- Resistance
May inhibit ps in mammalian mitochondria, which have ribosomes like bacteria
How do newly formed proteins know where to go?
For every organelle in the cell hay a specific class of signal sequence that determine where the new protein goes. These signal sequences bind to receptors which then mediates the import of the protein into the correct organelles
Draw and describe the nuclear pore complex.
The nuclear pore complex:
About 30 different proteins
500-1000 protein molecules
3000 to 4000 NPCs per mammalian cell
Mass: ~ 125 million daltons
How does the size of the protein molecule influence nuclear import?
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
How are larger proteins imported into the nucleus?
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 it cant anymore bind to the import receptor, so its free. This import receptor goes back to the cytoplasm to bind to a new cargo.
State the roles of mitochondria.
Respiration, ATP synthesis
Heat generation(in brown fat)
Fatty acid metabolism
Intermediary metabolism (synthesis & breakdown of biomolecules)
What are the 2 ways in which proteins end up in mitochondria?
Proteins are imported from the cytoplasm into mitochondria but mitochondria has its own DNA, which makes some of its proteins inside the organelle.
Describe protein import into the matrix of Mitochondria
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.
Describe Insertion of Proteins into the Inner Mitochondrial Membrane
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.
Describe transport of proteins onto the rough ER.
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.
Describe vesicular transport
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.
Give the signal sequences for the nucleus, ER and mitochondria.
Signal sequences determine protein localisation.
What is the Z helical form of DNA?
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…
Outline the Sanger sequencing method of DNA, and its advantages
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.
What are higher order DNA structures?
As well as different DNA sequences and different helical forms, there is 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
Describe the DNA of E coli
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
What does a 3’ 5’ link mean?
This refers to the carbon atoms of the sugar to which the oxygens on the phosphate are linked.
Describe the cell cycle in eukaryotes vs bacteria
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
Draw a diagram to demonstrate bacterial DNA replication
Hay 2 replication forks, where the parental DNA duplex unwinds to make the separated strands. This means that replication is bi directional in bacteria
How does replication initiate at eukaryotic cells?
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
What are the diff types of DNA polymerases?
Translesion polymerases are involved in allowing replication forks to bypass irreparable damage
Gamma polymerases= specific to mitochondrial DNA
How does DNA polymerase synthesise new DNA?
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
All known DNA polymerases add nucleotides to the 3′ OH end of the growing strand. Thus, DNA synthesis always proceeds in the 5′ to 3′ direction. Does this cause a problem?
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.
What are DNA replication inhibitors?
Clinically important drugs inhibit DNA synthesis
How do you enzymes facilitate reactions?
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
What are the two models that describe enzyme function?
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
Draw a graph and label it to explain enzyme kinetics.
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
How can this equation for the kinetics curve graph be rearranged to give a straight line graph?
What is competitive inhibition?
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.
What is non competitive inhibition?
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
what is allosteric regulation? Give an example using how CTP regulates the complex
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.
