Enzymes pt. 2 and 3 Flashcards
What is the reaction rate of a unimolecular irreversible first order reaction?
A -> P
v = k[A]
Thus the rate of P formation is directly proportional (rate constant k) to the concentration of A
What makes a reaction ‘first order’?
An exponent of 1
What is the reaction rate of a bimolecular irreversible second order reaction?
A + B ->
Rate of P formation is directly proportional (rate constant k) to the concentration of reactants
v = k[A][B]
What is the unit of the rate constant for a first order reaction?
s-1
reciprocal second
What is the unit of the rate constant for a second order reaction?
M-1 s-1
reciprocal molarity, reciprocal second
What is the rate of formation for a unimolecular reversible reaction?
A -> P k1 and P -> A k2
Rate of formation of P equals rate of P gain minus rate of P gain
v = k1[A] - k2[P]
Keq = ?
k1/k2 = [P]/[A]
What does a large Keq indicate?
Reaction favors products
What does a small Keq indicate?
Reaction favors reactants
What is the basic michaelis-menten equation?
E + S -> ES > E + P
and the reverse for rxn 1
ES = enzyme substrate complex
What are the units of the Michaelis constant?
M
What assumptions are made to simplify Michaelis-Menten?
[S]»_space;> [E]
and [ES] is relatively unchanging
No back reaction from pdt build up (k4 = 0)
Maximal velocity (rate of rxn) occurs when __________.
Enzyme is totally saturated with enzyme
Vmax = ?
= k3[E]total
What is the Michaelis-Menten equation?
Vo = Vm[S] / Km + [S]
What is Michaelis-Menten plotting?
Vo (rate) versus [S]
Km = ?
[S] at 1/2 Vmax
Filled sites = ?
Vo/Vm = [S] / Km+[S]
What is Km indicative of?
Enzyme-substrate affinity (higher Km, lower affinity)
In the special condition of k2»_space;> k3, what does that mean?
Making the product is the slowest reaction
Km = Kd ~ [E][S]/[ES]
A low Km means what?
High binding affinity
A high Km means what?
Low binding affinity
To judge affinity, what must be true?
k2 must be much greater than k3
Increasing your enzyme concentration will ________ your maximum velocity (reaction rate)
Increase
Turnover number = ? = ?
= k3 = kcat
“Catalytic ability” Typically 1-10^4
Higher value = higher catalytic ability
What is catalytic ability?
Number of S molecules converted to P by one E molecule in unit time under saturation conditions
(the best your enzyme can be functioning)
What does kcat / Km indicate?
The catalytic efficiency of how well an enzyme reacts with dilute amount of substrate
What type of enzymes have the higher kcat / Km values?
“Perfect” enzymes
What is the equation of a Lineweaver-Burk plot?
1/Vo = ( Km/Vm * 1/[S] ) + 1/Vm
What is the slope of a lineweaver-burk plot?
Km/Vm
The value of the slope in a lineweaver-burk plot is ______ proportional to the catalytic efficiency.
Inversely
What is the y intercept of a lineweaver-burk plot?
1/Vm
What is the x intercept of a lineweaver-burk plot?
-1/Km
What is the advantage of lineweaber-burk?
Provides easy-to-interpret visual display
What is the disadvantage of lineweaver-burk?
Distorts errors at low [S], compresses data at high [S]
What are the two main classes of two-substrate (or bi-substrate) reactions?
Sequential (ternary)
Ping-pong (double-displacement)
What is a sequential two substrate reaction? What are the kinds?
All substrates bind (ternary complex) before any product releases
Essentially, both come down before both come off.
Can be ordered (specific) or Random order
What is a ping-pong or double replacement mechanism?
For two-substrate reactions, this describe when one substrate binds and released product before the second substrate binds and releases product.
What enzyme is a classic ping pong enzyme?
Chymotrypsin - peptide bonds and releases product before water binds and releases water
What is an enzyme inhibitor?
A small molecule or ion that binds an enzyme and decreases its activity.
What are the two types of inhibitors?
- Reversible inhibitors
2. Irreversible inhibitors
What are the features of reversible inhibitors?
Bind the enzyme with noncovalent interactions
Dissociate rapidly
Allow the enzyme to recover its original activity
What are the features of irreversible inhibitors?
Bind the enzyme with covalent interactions
Result in permanent inactivation of the enzyme
What are competitive inhibitors?
Binds only to free enzyme and competes with the active site
- thus usually resembles the shape and structure of the substrate
- reduces number of ES complexes by taking up enzymes as part of their EI complexes
How do competitive inhibitors affect Vm?
They don’t!
How do competitive inhibitors affect Km?
Increases it! (i.e. decreases the apaprent binding affinity)
How do competitive inhibitors affect the catalytic efficiency?
Decrease!
Can the effects of competitive inhibitors be overcome?
Yes! By increasing the amount of substrate
When are competitive inhibitors most effective?
When substrate concentration is low
Many drugs are ______ or ________ analogs
Substrate or transition-state
Are substrate or transition-state analogs more potent inhibitors?
Transition-state because they bind more tightly
How do uncompetitive inhibitors work?
Bind to the ES complex NOT at the active site/enzyme alone
How do uncompetitive inhibitors affect Vm?
Decreases
How do uncompetitive inhibitors affect Km?
Decreases
How do uncompetitive inhibitors affect catalytic efficiency (kcat)?
No change
Can increasing substrate concentration overcome the effects of uncompetitive inhibition?
No
Do rate constants change in the presence of inhibitors?
NO! They just APPEAR to change (hence the term “apparent Km/Vm etc.”)
At very low substrate concentrations, are uncompetitive inhibitors successful?
No, but it becomes more and more powerful as you increase your substrate concentration
What is a noncompetitive inhibitor?
Binds to free enzymes or ES complex at a site different than the active site.
Hinders the reaction by distorting enzyme structure and preventing alignment of the catalytic center
How do noncompetitive inhibitors affect Vmax?
Decrease
How do noncompetitive inhibitors affect Km?
No change…the substrate doesnt really care if the inhibitor is there or not. Itll bind fine regardless
How do noncompetitive inhibitors affect catalytic efficiency (Kcat)?
Decrease
Does increasing the substrate concentration overcome the effects of noncompetitive inhibitors?
No
What common antibiotic is an example of a noncompetitive inhibitor?
Doxycycline
What type of inhibitor decreases both Vm AND Km?
Uncompetitive
How does allosteric control work?
Allosteric enzymes operate by positive cooperativity: binding of substrate to one active site increases activity of other active sites.
What effect do allosetic enzymes have on Km?
Usually decreases with increasing substrate concentration
How do binding curves of allosteric enzymes appear?
S-shaped
Allosteric enzymes are typically ______-______ enzymes
Rate-determining
What is allosteric control?
Noncovalent binding of a ligand (effector) to a site other than the active site, thereby affecting the activity of the active site
What is a homotropic effector?
Effector is the same as the substrate
Site is usually adjacent to active site
Interaction almost always increases activity
What is heterotropic effector?
An effector that is the different than the substrate
Usually distant from the active site
Can increase or decrease activity
Allosteric control operates by effector- induced conformational changes that alter activity by changing ___, ____, or both.
Km (K class) or Vm (V class)
Are allosteric enzymes generally oligomers with multiple subunits and active sites?
Yes
How is ATCase affected by allosteric control?
Aspartate will bind to the enzyme when in the tense state, pushing it into the relaxed state and increasing the reaction rate in an exponential way (S-shaped curve)
OR
CTP can also bind and stabilize the tense state, pushing the curve to the right
How is PKA an example of allosteric regulation?
Examples of protein binding increasing or decreasing activity:
2 subunits are regulatory
2 subunits are catalytic
cAMP binds to the active sites and pulls the regulator units away, allowing PKA to become active
How do reversible covalent modifications affect enzyme activity?
Charged groups such as phosphate, sulfate, or acetate, can bind enzymes and cause conformational changes that alter function.
What is the most common form of reversible covalent modifications to regulate enzyme activity?
Phosphorylation/dephosphorylation
via kinases and phosphatases
Each step is considered IRREVERSIBLE even if the process as a whole is reversible (i.e. there are 2 different enzymes/2 different reactions to complete the cycle).
