Metabolism 2.5, 2.6, 7.1, 8.1 Flashcards
What is the role on an enzyme?
> speeding up the reaction (catalysts) - no change in reaction - new reaction pathway > temperature could be increased - biological systems are sensitive
What is active site?
- enzymes are proteins
- made often
- 3D structure
- structure determines function
- active site - region where the substrate binds
- enzyme-substrate complex
What are different models of enzymes?
> lock and key - exact fit between enzyme and substrate - 1 enzyme = 1 substrate - products have different shape - formed an released > induced fit model - proteins can change their shape - substrate binds - induces change in enzyme - active site reshaped - 1 enzyme = many substrates (similar types)
How does an enzyme convert substrate to a product?
- breakdown
- substrates bind to active site
- specific bonds exposed
- stretched (increases likelihood of being broken)
- specific bonds exposed
- substrates bind to active site
- synthesis
- substrates in active site are positioned
- more likely collision
- substrates in active site are positioned
How do substrates bind to active site?
- collision
- particles are in motion
- substrates are smaller so they move faster
What are the factors affecting enzyme activity
> enzyme and substrate concentration
- the more enzyme, the faster
- substrate concentration reaches saturation - fixed amount of enzymes
temperature
- the higher, the higher kinetic energy
- too high, enzyme denaturation
pH
- for each reaction there is an optimal pH
- different pH lows the reaction (or prohibits)
What is denaturation?
> high temperature or high / low pH - active site is altered - no catalyst > homeostasis of cells is in danger - environment is not suitable
What is metabolism?
> sum of all reactions that occur in organism to maintain life > metabolic pathways - sequence of small steps - chain of reactions - sometimes cycle
What are types of inhibitors?
- inhibitors reduce enzyme activity
- competitive inhibitor
- similar to substrate
- active site
- competes
- non-competitive inhibition
- different structure
- binds to enzyme in different way
- changes shape of enzyme
- examples (drugs)
- saquinavir - HIV protease inhibitor
- nevirapine - HIV reverse transcriptase inhibitor
How is metabolic pathway regulated?
- end-product inhibition
- end product of the pathway inhibits metabolism
- example
- threonine (initial substrate) binds to theorinine deaminase
- end product → isoleucine
- as the concentration increases it binds to allosteric site (non-competitive inhibition)
What are immobilised enzymes?
- enzymes attached to other material (glass, alginate gel)
- enzyme movement restricted
- advantages
- separated from products, stopping reaction at ideal time
- recycling (cost savings)
- stability of enzyme increases
- changes in temp or pH
- higher concentration of enzyme than dissolved enzyme
Why is lactose-free milk produced?
- lactose → glucose + galactose
- why?
- for people who are lactose intolerant
- glucose and galactose are sweeter
- less sugar in milk products
- lactose crystallises during ice cream production
- quicker fermentation (cheese)
Hershey-Chase experiment
- aim: testing whether proteins or DNA is genetic material
- viruses injected genetic material
- radioactive S-35 for proteins
- radioactive P-32 for DNA
- infecting bacteria
- blender to separate non-genetic and genetic material
- measured radioactivity in supernatant (substance outside) or pellet (genetic material)
- P-32 in pellet
What is a nucleotide?
- sugar
- pentose (or different)
- nitrogenous base
- 4 types
- phosphate group
- formed by forming covalent bonds between phosphate group and sugar
What is the structure of DNA?
- double helix
- 2 strands of nucleotides linked by covalent bonds
- different directions
- complementary base pairing
- A with T
- C with G
What are the differences between RNA and DNA?
- different sugars
- DNA has less oxygen
- 2 polymers of nucleotides in DNA
- T → U
What is semi-conservative replication?
- base sequence on template determines the sequence on new strand
- 1 strand parent, 1 strand new
Meselson and Stahl experiment
- bacteria grown in N-15 (heavy) medium
- transferred to N-14
- samples after 0, 20 and 40 minutes
- 0 min: heavy and light
- 20 min (first generation): all intermediate
- second generation: half intermediate, half light
= semi-conservative replication
What is the first stage of DNA replication?
- helicase unwinds double helix
- breaking H-bonds
- group of enzymes
- use ATP
- forming replication fork
- leading strand = towards replication fork
- lagging strand = opposite
What is the second step of DNA replication?
- DNA polymerase III
- elongates nuclear acid
- attaches new nucleotides to a stand
- attaches to 3’ end
- starts building the chain from 5’
- RNA primers
- synthesised by primase
- start point
- leading has 1
- lagging has many = Okazaki fragments
- DNA strands rewind
What is the function of DNA polymerase?
- DNA polymerase III
- replication in E. coli
- check
- polymerases copy bases, check it and cut out if wrong
- adding nucleotide
- unless H-bonds are formed, nucleotide is discarded
- nucleotides are linked by covalent bonds
- adds to 3’ end
- DNA polymerase I
- repair
- exchange RNA primers into DNA
What is the role of nucleosomes?
- eukaryotic DNA in form of chromosomes
- wrapped around histones
- 8 histones + DNA = nucleosomes
- chromosomes can coil
- packing DNA
What is the function of DNA primase?
- generates short RNA primer
- one on leading strand and maybe on lagging strand
- extension of nucleotide chain
- RNA removed by DNA polymerase I (or exonuclease)
What is the function of DNA ligase?
- joining Okazaki fragments
- covalent bonds between sugar and phosphate
- repairs damaged DNA
What is the function of topoisomerase?
- helicase unwinds DNA
- creates tension
- makes a cut in one strand
- holds to the spot
- helix spins (no overwinding)
- reconnects the spot
- in E. coli —> DNA gyrase
What is a leading and a lagging strand?
- strands in anti-parallel
- leading strand follows the fork
What is the direction of replication?
- from 5’ to 3’
- starting with sugar ending with phosphate group
What is the function of non-coding regions of DNA?
- satellite DNA
- components of heterochromatin and centromeres
- telomeres
- repetitive regions at the end of chromosome
- protection
- introns
- within genes
- removed in RNA splicing
- non-coding RNA genes
- not translated into protein
- genes for tRNA
- gene regulatory sequences
- transcription
- promoters, enhancers and silencers
What are the steps of PCR?
- polymerase chain reaction
- denaturation
- heated to 95ºC
- separation
- annealing
- cooled to 55ºC
- primers anneal
- elongation
- 72ºC
- Taq polymerase
- the cycle is repeated several time to obtain many copies of DNA
What is the role of Taq polymerase in PCR?
- DNA polymerase that is heat-resistant
What is DNA sequencing?
> unknown DNA in tube + enzymes necessary for replication
dideoxyribonucleotides are added
- fluorescent
- attach to bases but after them no new bases added
fragments separated by length
- electrophoresis
colour and length of fragments compared —> sequence
