List all factors affecting enzyme activity.
Temperature, pH, [enzyme], [substrate], competitive and non-competitive inhibition.
How does temperature affect enzyme activity?
- Increased T = molecules have a higher KE.
- More successful collisions between enzymes and substrate per unit time - more E-S complexes formed per unit time.
- However, excessive vibration from high temperatures (usually 60+ degrees) can break bonds holding the enzyme’s tertiary structure together - denatured.
How does pH affect enzyme activity?
- Change in pH alters the charges on the amino acids that make up the enzyme’s active site - no E-S complexes can be formed as substrate can’t attach.
- Could cause the bonds maintaining the tertiary structure to break - i.e. interferes with the ionic bonds etc. in tertiary structure. This alters the shape of the active site and therefore prevents E-S complexes from forming.
How does [enzyme] affect enzyme activity?
- Increase in [enzyme] = increase in rate as more E-S complexes formed per unit time - more likely collisions between enzymes and their substrate per unit time.
- But if [substrate] is limited, there are enough active sites to accommodate all the available substrate molecules - no further increase in rate.
How does [substrate] affect enzyme activity?
Assuming a fixed [enzyme]:
- [Substrate] is steadily increased, the rate increased in proportion initially - limited number of substrate molecules for enzymes.
- More substrate added until Vmax (max rate) is reached - all active sites are full so adding more makes no difference.
- [Substrate] decreases with time, so rate also decreases with time - therefore the initial rate is the highest rate during a reaction.
- Rate levels off if substrate in excess.
NB = On a volume of x produced by y enzyme/time graph, if line tails off, it is likely because all substrate used up.
Describe competitive enzyme inhibition.
- Inhibitor has similiar structure to the substrate complementary to the enzyme.
- Occupies the enzyme’s active site, competing with the substrate molecules.
- This prevents E-S complexes from being formed.
NB = the relative [substrate]/[inhibitor] determines how much inhibition
i. e. high [inhibitor] = little substrate reaches enzyme active site.
i. e. high [substrate] = will increase chances of substrate reaching enzyme active site and increases rate of inhibited reaction, up to a certain point.
Describe non-competitive inhibition.
- Inhibitors bind elsewhere on the enzyme away from the active site.
- Enzyme shape and therefore active site shape changes.
- Prevents E-S complexes from forming as substrate cannot bind.
- Increase in [substrate] won’t make any significant difference.
How can we see if an enzyme-controlled reaction is being inhibited by competitive or non-competitive inhibition?
Increase the [substrate] and observe rate.
If rate increases, competitive inhibition.
If rate stays virtually constant, non-competitive inhibition.
Describe end-product enzyme inhibition (+).
Enzymes at the start of a metabolic pathway are inhibited more or less by the product of the pathway.
Basically a system of negative feedback - high product concentration - less needed so enzyme A at start of pathway inhibited so less produced and works vice versa.
Usually non-competitive inhibition.
=> Ensures an almost constant [product].
Functions of DNA and RNA - generally.
DNA - holds genetic information, coding for proteins. Long polynucleotide chain.
RNA - transfers genetic information from DNA to the ribosomes. Relatively short polynucleotide chain.
DNA nucleotide structure.
RNA nucleotide structure.
Deoxyribose 5C sugar, bonded to an organic nitrogenous base on C1 and to a phosphate group on C4.
Ribose 5C sugar, bonded to an organic nitrogenous base (T replaced by U) on C1 and to a phosphate group on C4.
Importance of condensation reactions in forming DNA.
Bonds holding nucleotide together are formed by condensation reactions.
Phosphodiester bonds (bonds between individual nucleotides - C3 on sugar and phosphate) formed by condensation reactions.
Complementary base pairing occurs between which nitrogenous bases? How many H-bonds?
Adenine – Thymine. 2 x H-bonds.
Cytosine —Guanine. 3 x H-bonds
DNA is in what form? Why is it a stable molecule?
Long polynucleotide chain coiled into a double helix due to hydrogen bonding between the bases. Antiparallel polynucleotide strands run in opposite directions.
Stable because:
- Phosphodiester backbone protects the more chemically reactive organic bases inside the double helix.
- MANY hydrogen bonds link the organic base pairs, adding more stability. More C—G pairing leads to a more stable DNA molecule, as more H-bonds.
DNA’s structure-function relationship.
- Stable, hereditary structure which rarely mutates.
- 2 separate DNA strands joined by H-bonds so can be separated in DNA replication and protein synthesis.
- Extremely large molecule - holds lots of genetic information.
- Base pairs within the helical cylinder - largely protected by external chemical and physical forces.
- Base pairing leads to DNA being able to replicate and to transfer information as mRNA.
Comment on DNA’s simplicity.
The relative simplicity of DNA led many scientists to doubt that it carried the genetic code.
Comment on 5’ to 3’ direction.
Two strands are antiparallel as one runs in the 5’ (C5) to 3’ (C3) direction.
DNA polymerase can only add new nucleotides to 3’ C3 (has an -OH group - condensation reactions).
Therefore, new nucleotides can only be added in the 5’ to 3’ direction as it needs to attach onto 3’ carbon, so if in 3’ to 5’ direction, wouldn’t work as would need to add to 5’ C.
Why is DNA replication described as being semi-conservative?
Each of the new DNA molecules contains one of the original DNA strands - half of the original DNA molecule is present in each new DNA molecule.
Describe the process of semi-conservative DNA replication.
- DNA helicase enzyme breaks the H-bonds linking the base pairs of DNA.
- Double helix separates into two strands and unwinds.
- Each exposed polynucleotide strand then acts as a template to which complementary free nucleotides bind by specific, complementary base pairing.
- DNA polymerase joins together polynucleotide strand, as each of the original DNA polynucleotide strands
- Each of the new DNA molecules contains one of the original DNA strands.
NB - a source of chemical energy is required to drive the process.
Suggest evidence for the Watson-Crick model of semi-conservative replication.
- Bacteria with 15N placed in 14N medium so no new 15N available.
- After one generation, one 15N/14N mixed strand. (1 double strand).
- After two generations, one 15N/14N mixed strand and one 14N molecule. (2 double strands).
- After three generations, one 15N/14N mixed strand and three 14N molecules (4 double strands).
What is ATP? Uses and reasons?
- Adenosine Triphosphate.
- Phosphorylated macromolecule - nucleotide derivative.
- Used as an immediate energy source to carry out processes in cells.
- Can store energy because of the unstable bonds between the phosphate groups. These bonds release a considerable amount of energy while being relatively easy to break and having a low activation energy.
- NOT a good long term energy store as very unstable.
ATP synthesis/hydrolysis reactions?
ATP hydrolysed to ADP + Pi by ATP hydrolase enzyme.
ATP synthesised from ADP + Pi by ATP synthase in a condensation reaction.
In which processes is ATP synthesised?
- Photophosphorylation in plants during in photosynthesis.
- Oxidative phosphorylation in animals and plants during respiration.
- Substrate-level phosphorylation in plant and animal cells - donor molecules donate Pi to ADP. Occurs during Krebs Cycle?
Roles of ATP?
- Immediate source of energy for a cell; better immediate source of energy than glucose because:
- energy is in more manageable quantities as energy from 1 ATP molecule is less than energy from 1 Glucose molecule.
- hydrolysis of ATP -> ADP + Pi is a single reaction whereas glycolysis takes longer as it is a series of reactions. - ATP hydrolysis coupled to other energy-requiring processes/reactions in cells.
- ATP provides energy needed to build up macromolecules from their basic units, such as polypeptides from amino acids.
- Muscle contraction - energy provided for muscle fibres to slide past one another and to therefore shorten length of muscle fibre.
- Active transport - ATP provides energy to change shape of carrier proteins in plasma membranes so molecules/ions can be moved against a concentration gradient.
- Secretion - ATP needed to form the lysosomes necessary for the secretion of cell products, such as insulin from pancreas/plant auxins.
- ATP is also used to phosphorylate other compounds - which lowers the Ea of enzyme-catalysed reactions -> phosphorylation of glucose molecules at the start of glycolysis.
