Enzymes Part 2 (Kinetics) Flashcards
Endergonic Reaction
- ΔG > 0
- Non-spontaneous; requires input of energy
- Products have higher free energy than the reactants
Exergonic Reaction
- ΔG < 0
- Spontaneous
How can an endergonic reaction be driven to move forward?
By coupling it with an exergonic (favorable) reaction
Main molecule utilized in enzyme coupled reactions:
ATP
- adenine (nitrogenous base)
- ribose (5 carbon sugar)
- three phosphate groups
- -3.5 total charge
ATP has a high energy phosphate bond. Removal of 1 phosphate group leads to ADP and gives off a considerable amount of energy.
What type of reaction occurs and how much energy is given off?
- hydrolysis (a phosphate bond is cleaved)
- ΔG = -30.5 kJ/mol
ATP + H2O → ADP + Pi
ΔG =
-30.5 kJ/mol
ADP + Pi → ATP
ΔG =
+30.5 kJ/mol
What is the molecular basis for the large amount of energy given of by the conversion of
ATP + H2O → ADP + Pi
- Electrostatic repulsion between the three phosphates destabilizes ATP molecule.
- ATP has -3.5 charge
- ADP has -2.5 charge
- After hydrolysis, an individual phosphate can be stabilized by resonance stabilization, which makes it more stable than its form in ATP.
Equilibrium is a state of:
maximum stability
A process is spontaneous and can perform work only when it is:
moving toward equilibrium
Reaction velocity versus [E]:
- more enzyme = faster rate.
- linear correlation
- substrate has more enzyme to bind to, increases [ES], increases product formation.
Reaction velocity versus [S]:
- rate increases asymptomatically with increasing [S]
- Velocity initially increases linearly, but then stabilizes and becomes constant when all enzyme active sites are saturated.
If you want to increase Vmax, you need to increase:
[E]
E + S ⇔ ES ⇒ E + P
What is Km?
- Michaelis Constant
- Conversion of E + S ⇔ ES
- reflects affinity for a substrate to an enzyme
- Km = [S] at ½Vmax
E + S ⇔ ES ⇒ E + P
What is Kcat?
- Rate of ES ⇒ E + P
- (Kcat)([E]) = Vmax
(Kcat)([E]) =
Vmax
Michaelis Menten Equation
- v = initial velocity at a given [S]
½Vmax =
Km
Lineweaver-Burk Plot Equation:
- x-intercept = -1/Km
- y-intercept = 1/Vmax
Km only changes with:
pH and temperature
Does not change with [E] or [S]
Numerically, Km =
[S] at ½Vmax
Smaller the Km:
- higher affinity of enzyme for substrate
- “tighter ES binding”
Larger the Km:
- lower affinity of enzyme for substrate
- “less tight ES binding”
Vmax is linearly dependent on:
[E]
Kcat only changes with:
- pH and temperature
- does not change with [E] or [S]
- a constant
Conceptually, Vmax is:
maximum velocity with which the enzyme can catalyze the reaction
Conceptually, Kcat is:
- kcat = (Vmax)([E])
- turnover number of an enzyme reflecting the number of moles of substrates converted to products per sec per mol of enzyme
- ES converted to E + P
Higher the Kcat:
- more product produced per second per mole of enzyme
- “high turnover:
Fastest way to regulate enzyme activity:
- phosphorylation/dephosphorylation of the enzyme
- most phosphorylation occurs on the serine, threonine, and tyrosine amino acids of an enzyme (all have -OH groups)
Slowest way to regulate enzyme activity:
extracellular signalling
Enzyme regulation by specific proteolysis:
- irreversible
The two types of enzyme inhibitors:
- irreversible
- reversible
- competitive
- uncompetitive
- noncompetitive
Irreversible inhibitors are:
- molecules that covalently bind to an active site amino acid of the enzyme to inhibit the activity.
- substrate analogs
- Examples:
- penicillin
- sarin (nerve gas)
- aspirin
Reversible inhibitors are:
- molecules that bind reversibly to inhibit enzyme activity
- Three kinds:
- competitive
- uncompetitive
- noncompetitive
Competitive inhibitors:
- reversible
- bind to active site of enzyme, compete with the substrate for active site slots.
- Km INCREASED, Vmax UNCHANGED
- can be overcome by increasing [S]
Noncompetitive inhibitors:
- reversible
- bind to a separate site of the enzyme (not the active site)
- Km UNCHANGE, Vmax DECREASED
- increasing [S] does not help since their is no competition for the active site
Example of a competitive inhibitor:
- statins
- inhibit enzyme involved in cholesterol synthesis
Transition State Analogs:
- potent inhibitors
- stable molecules that resemble geometric and/or electronic features of the highly unstable transition state
- binding is often much tighter than the substrate because they fit all elements of the active site
Allosteric Enzymes:
- have two binding sites:
- active site (for substrate)
- allosteric site (for a noncompetitive inhibitor or effector)
- all are oligomeric (>1 peptide sequence)
- allosteric molecules do not resemble substrates
V versus [S] curve of allosteric enzymes:
- sigmoidal (S-shaped)
- due to cooperative substrate binding
The two equilibrium states of allosteric enzymes:
- R-state (active; strong substrate binding)
- T-state (inactive; weak substrate binding)
Allosteric activators stabilize:
- the R-state (active) of an allosteric enzyme
- increases substrate binding
- increases activity
Allosteric inhibitors stabilize:
- the T-state (inactive) of an allosteric enzyme
- decreases substrate binding
- decreases activity
K-type Allosteric Inhibitor:
- increase K0.5 (DECREASES AFFINITY)
- no effect on Vmax
K-type Allosteric Activator:
- decreases K0.5 (INCREASES AFFINITY)
- no effect on Vmax
V-type Allosteric Inhibitor:
- decreases Vmax
- no effect on K0.5
- no effect on affinity
V-type Allosteric Activator:
- increases Vmax
- no effect on K0.5
- no effect on affinity
