Enzymes and Metabolism
What does delta G being positive means? Delta G=negative?
-DeltaG being positive means the reaction is spontaneous, ends with less free energy, and loses that energy to the surroundings
-DeltaG being negative means reaction is not spontaneous, ends with more free energy, and gains energy from the surroundings
Why is supercoiling of DNA effective?
Helps us store more genetic information in smaller space. Think about how small a bacteria is.
Why does the electron transport chain need oxygen? What happens when we have no oxygen?
We don't have the final electron acceptor, O2, anymore. So the concentration gradient doesn't get properly built, which means electron transport chain cannot work.
What are cellular membranes made out of? How does this help their semi-permeable function?
Phospholipid bilayers. The heads are polar and the tails are nonpolar, making it so only very small, nonpolar things (or water) can pass freely.
What does a DNA polymerase do? On what side does it attach? What functional group does it need to attach?
DNA polymerase attahced to 3' end and needs an OH group to attach.
What is energy coupling? Why is it often necessary? Give an example about a process that we've talked about that used energy coupling.
When an endergonic reaction occurs with an exergonic reaction, so energy can be supplied to the endergonic reaction from the exergonic reaction.
Ex: phosphates hydrolyze off nucleotides to synthesize nucleic acids
What does the endosymbiotic theory state?
-larger prokaryotic cell engulfed a proteobacterium, the proteobacterium 'enjoyed' sheltered protection of the larger cell and the supply of nutrients, while the larger cell 'enjoyed' xtra ATP molecules that leaked out of the proteobacterium, it was mutually beneficial, they became obligate endosymbiont who needed each other, believed to be how we came to eukaryotic cells today
What happens to the citric acid cycle when there's no oxygen? Why?
Because the ETC no longer works, there is a buildup of NADH. NADH is an inhibitor of the citric acid cycle, so the citric acid cycle no longer functions. We can only do glycolysis when there's no oxygen.
Compare and contrast facilitated diffusion and active transport.
Facilitated diffusion is when there are protein channels available for molecules to passively diffuse in/out through osmosis, no energy is needed.
Active transport also has protein channels, but energy is needed.
Name the three steps of Kunkel Mutagenesis. Give a short description of each.
Phosphorylation = phosphorylate 33 bp oligo beforehand with kinase
Annealing = Heat up ssDNA and oligo to denature it, they anneal/bind (except for the 3 bp mutation)
Polymerization = DNA polymerase comes in and adds nucleotides until its' complete, then polynucleotide kinase changes OH group to phosphate, and ligase seals it up
What's the difference between competitive inhibitors and allosteric regulators? If something is inhibited only at high concentrations, what type of inhibitor does that usually indicate?
-Competitive inhibitors= resembled natural substrate and binds to active site
-Allosteric regulators/non-competitive inhibitors = bind to a separate site away from active site, can activate or inhibit enzyme
-Usually indicates a competitive inhibitor, bc it'll have to compete with actual substrate before it can inhibit
Name three pieces of molecular evidence for the endosymbiotic theory for chloroplasts or/and mitochondria.
-mitochondria/chloroplasts both have their own double-stranded, circular, supercoiled DNA genomes like prokaryotic cells
-mitochondria/chloroplasts can divide on their own through binary fission like prokaryotic cells
-mitochondria/chloroplasts both have highly convoluted membrane structure that maximizes surface area and energy processes, which is similar to what aerobic bacteria/cyanobacteria have
How is lactic acid fermentation a regenerative process?
The reduction of two pyruvate to 2 lactate is linked to the oxidation process of NADH turning back to NAD+, providing the NAD+/coenzyme needed for glycolysis to be able to occur again.
What are active transport proteins sometimes used for? Give an example.
Active transport proteins are sometimes used to establish concentration gradients. Ex: Establishing the H+ concentration gradient in the ETC.
What did UC Davis give us to start our lab? (Hint: 2 things)
- single stranded BgIB plasmid with antibiotic resistance gene, WT BgB gene, lac gene, T7 promoter
- 33 base pair oligonucleotide that has our 3 bp mutation in it and the rest 30 are wildtype
The final rxn of glycolysis catalyzes the hydrolysis of a phosphate from phosphoenolpyruvate yielding pyruvate, an exergonic rxn with a deltaG of -61.9 kJ/mol. This rxn is coupled to the synthesis of ATP from ADP and phosphate. The deltaG of hydrolysis of ATP to ADP and phosphate is -30.5 kJ mol.
Based on this info, what is the total deltaG of this coupled rxn, and is it exergonic or endergonic?
Total deltaG of coupled rxn is -31.4 kJ/mol, and it overall exergonic.
List the functions of microtubules, microfilaments, and intermediate filaments.
-Microtubules = cellular highways/transport tracks where vesicles are moved by motor proteins, made of globular tubulin
-Microfilaments = help w structural support, can move and be assembled or disassembled, drive changes in cell shape or movement (motility), ex: muscle cell contraction
-Intermediate filaments = help with structure, support, tether organelles, made of fibrous proteins like keratin
Why is phosphofructokinase an ideal inhibitor in glycolysis?
When ATP concentrations are high: Glucose can still get trapped by hexokinase, so we can store it. But our body won't have phosphofructokinase (since we can inhibit it) oxidize the sugar if we don't need to, so it won't get used up if we don't need it.
What happened when we put our double-stranded oligos into bacteria? Why? What was our end product?
The bacteria sensed uracil in the DNA plasmid, and cut out the uracil, effectively deleting that strand. A new strand could then be synthesized from the remaining strand. We ended with a complete double stranded oligonucleotide that was normal (no uracil) and had our 3 base pair mutation.
During exercise, your muscles make more AMP as ATP is used up.
The enzyme phosphofructokinase-1 (PFK-1) helps break down glucose by turning fructose-6-phosphate into fructose-1,6-bisphosphate.
Scientists find that:
At low substrate levels, PFK-1 works slowly.
Once substrate concentration gets high enough, its activity suddenly increases.
Adding AMP makes this sharp increase happen at lower substrate levels.
PFK-1 shows positive cooperativity — when one substrate binds, it makes other subunits bind substrate more easily (higher affinity).
AMP is an allosteric activator that keeps the enzyme in its high-affinity form, so it becomes active at lower substrate concentrations.
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Name the steps of how we get from a hydrolase gene to a fully functioning protein we can use.
DNA transcribed to mRNA -> cytoplasmic ribosome translates to synthesize protein (ribosome will move along mRNA as tRNA molecules will deliver correct amino acids specified by mRNA) -> protein folding -> glycosylation(adding carb groups)/phosphorylation modifications to protein to help direct it to destination -> transported to final destination
How does ATP only inhibit PFK at high concentrations, even tho its not a competitive inhibitor?
PFK (phosphofructokinase) has two ATP binding sites, allosteric site and activie site
Naturally, the allosteric site has low affinity for ATP, and the active site has high affinity for ATP. If there's a lot of ATP, it'll bind to the allosteric site, induce a shape change, and the active site will change to have a low affinity for ATP, effectively inhibiting enzyme. (*ATP can't help phosphorylate sugar now, so reaction won't happen)