Enzyme Regulation Flashcards
What are 3 ways to regulate enzymes?
- Change the amount of enzyme available
- Have the enzyme interact with ligands (inhibitors, activators)
- Have the enzyme covalently bind with modification molecules
What is mass action as it relates to enzymatic activity?
Mass action means working through concentration gradients. If you increase the concentration of a competitive inhibitor it will eventually steal interaction opportunities from the more ideally “fit” substrate
What are the 3 types of inhibition? Describe and draw
- Competitive inhibition - substrate homologue sneaks in and interacts in the place of the substrate. Changes Km but not Vmax
- Noncompetitive inhibition - inhibitor binds to allosteric site and decreases the functionality of the enzyme. Vmax decreases but Km stays the same (usually, except for v-type noncompetitive inhibitors)
- uncompetitive inhibition - inhibitor AND substrate bind, but inhibitor keeps substrate stuck to enzyme and doesn’t allow the reaction to carry forward.
What is enzymatic efficiency?
Kcat/Km
What does Km represent?
Binding affinity. How easy it is for substrate to bind to enzyme
What does Kcat represent?
Turnover speed. How quickly the enzyme cranks out products
What does allosteric mean?
An allosteric enzyme has at least 2 active states (off, inactive, active). Active state binds effectively, inactive state still binds, but just not well.
Where does competitive inhibition occur?
At the catalytic site
What type of inhibition occurs at the catalytic site?
competitive inhibition
Where does noncompetitive inhibition occur?
At the allosteric site
What are some examples of covalent modification of a protein?
Phosphorylation, adenylylation. These are usually caused by some hormone-mediated signalling cascade
What is the timeframe that enzyme regulation occurs within?
- modifying quantity of enzyme
- modifying activity of current enzyme
- hours to days (you need to decrease gene expression) or minutes (if you induce post-translation protein modification)
- nearly instantaneously if you’re trying to use binding ligands to affect change
What does kinase do?
Adds phosphates
What does phosphatase do?
Takes away phosphates
Where does the control for signal cascades occur?
Usually at the first signal. If you modify just the first signal you only need to change the concentration of one binding molecule. But if you change later on you might have to make multiple adjustments. First is simplest, and first protein often has surface area exposed to extracellular matrix
What type of inhibition increases Km but does not impact Vmax
Competitive (once you outcompete inhibitors the efficiency is the same)
What type of inhibition decreases Vmax without impacting Km?
Noncompetitive (substrate concentration doesn’t matter)
What does a michaelis menten graph look like? What are its axes? Draw
Velocity vs. substrate concentration, an asymptotic curve.
What does a lineweaver burk plot look like? What are its axes? What is its x-intercept? What about the y-intercept?
Lineweaver burk plot is 1/v vs. 1/s. linear. x intercept is -1/km y intercept is 1/vmax
How does competitive inhibition change lineweaver burk plot?
y-intercept is constant, slope gets steeper with more inhibitor, x-intercept approaches 0
What type of inhibition causes the lineweaver burk plot to have a flatter slope and a change in the y-intercept?
noncompetitive inhibition (changes vmax but not km(
How does noncompetitive inhibition change the lineweaver burk plot?
make slope steeper, vmax decreases
What constants matter in competitive inhibition?
Km vs. Ki. Ki is basically binding affinity of inhibitor. Km is binding affinity of attacking protein.
Which type of inhibition is more efficient?
Noncompetitive. Well actually suicide inhibition is better but super rare. Suicide inhibition is when an inhibitor binds permanently to the enzyme
What are positive and/or negative feedback loops for enzyme regulation?
as more product is made, the product is actually used to regulate further production. positive feedback increases production, negative feedback slows production
Draw a michaelis menten plot for an allosteric enzyme
changing binding affinities make an S shape (low affinity when low S, high affinity when high S)
Draw a normal michaelis menten plot and label axis
Hill looking function (high affinity at top right)
Draw a lineweaver burk plot
flat line
Draw a lineweaver burk plot for an allosteric enzyme
looks like the right half of a U (high affinity at bottom left)
What causes an allosteric enzyme to change its binding affinity?
Something, whether a substrate or supporting molecule, interacts with the enzyme (often at least a dimer) and either induces shape change right away to the whole enzyme or induces shape change wherever the substrate bound.
What is concerted binding for enzymes?
Conformational shape change occurs for both dimers at the same time (as soon as first substrate binds), and then second substrate binds to newly changed dimer. concerted means simultaneous shape change. Both dimer halves immediately jump from T (low binding affinity state) to R (high binding affinity state)
What does sequential conformation adjustment in enzymes look like?
One half of dimer binds substrate and changes shape, then the next half binds and changes shape. Not simultaneous. Takes both for dimer to get into R (high binding affinity state)
What does an allosteric lineweaver burk plot look like?
The lines have curved (upwards) slopes, because as less substrate is present/bound more and more of the enzyme are found in the inefficient conformation
What are k-type inhibitors?
K-type inhibitors are allosteric inhibitors that only change Km as they switch conformations (binding affinity increases)
What are v-type inhibitors?
V-type inhibitors change vmax and km as they change conformation. V-type enzymes (if meant to be positive) could be rapid response enzymes, maybe for use in tissue damage.
What are G proteins used for?
Signalling cascade from extracellular matrix to (usually) gene expression
What is an example of a zymogen?
Prothrombin, (turns into thrombin, which is forced into fibrin, which eventually impact pathways. Digestion enzymes in the liver
What is an isozyme?
“isomer” of protein enzyme, slightly altered from initial enzyme.
What type of enzyme is a blood clotting enzyme?
v-type enzyme: extremely fast response so highly active state means vmax increases and km decreases
Are isozymes good or bad?
Some isozymes are great! They represent innovation and diversity in the body. But isozymes can also represent tissue damage.
Anabolic vs. Catabolic?
Anabolic means synthesize
Catabolic means breakdown
